P=0.0088). Of 25 patients who changed medication due to therapy-related adverse effects, 20 (80%) patients fully completed the therapy after drug change. In adjuvant endocrine therapy, a lowering of the non-adherence rate to 10.8%, the lowest reported in the literature, is realistic when patients are cared for by a specialised oncologic unit focusing on the individual needs of the patients.Previous research evaluating the use of adjuvant endocrine therapy among postmenopausal breast cancer patients showed with 15–50% wide ranges of non-adherence rates. We evaluated this issue by analysing an unselected study group comprising of 325 postmenopausal women, diagnosed from 1997 to 2003 with hormonal receptor-positive invasive breast cancer. The different clinical situations that led to the discontinuation of adjuvant endocrine therapy were clearly defined and differentiated: non-adherence was not simply the act of stopping medication, but rather the manifestation of an intentional behaviour of the patient. Of the 287 patients who initiated endocrine therapy, 191 (66.6%) fully completed this treatment. Thirty-one patients (10.8%) showed non-adherence to therapy. Patients who had follow-up with a general practitioner, rather than in an oncologic unit, were more likely to be non-adherent ( Since the late 1990s, adjuvant systemic endocrine therapy was recommended for the vast majority of postmenopausal patients with hormonal receptor (HR)-positive breast carcinomas. The sixth St Gallen International Consensus Panel on the Treatment of Primary Breast Cancer refined these guidelines in 1998: with the exception of low-risk, node-negative patients, the panel recommended a full 5 years of tamoxifen therapy for all elderly women with HR-positive breast cancer  are important, but are not the only contributors. Both factors were evaluated in several studies, in clinical trials , whose carcinomas were HR–positive, and therefore were candidates for adjuvant endocrine therapy. We define and evaluate the diverse clinical situations that hinder the realisation of the target of a complete 5-year therapy.Data regarding all postmenopausal non-metastatic breast cancer patients, who received surgical therapy between 1997 and 2003 at the Department of Gynaecology and Obstetrics of the University Hospital Basel , form the basis of the current analysis. Of these 393 patients, 325 had HR-positive carcinomas (82.7%) and 68 had HR-negative tumours (17.3%). The 325 patients with HR-positive carcinomas were evaluated in this study. We had complete follow-up in 323 of these patients, whereas two patients were lost to follow-up, one after 3 months and the other after 12 months.The following data were collected from the medical records and were available for all patients: age at initial diagnosis, tumour stage according to the American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) TNM Classification, histologic subtype, grading, oestrogen receptor (ER) and progesterone receptor (PR) status, surgery type, and receipt of adjuvant chemotherapy and/or radiation. HER-2/neu status was available for 273 patients (84.0%).The treatment recommendations for all patients were based on the decision of the interdisciplinary tumour board of the University Hospital Basel. Since 1997, all HR-positive patients, with few exceptions, were recommended to have adjuvant endocrine therapy. All patients received a comprehensive consultation at the departmental oncology unit during which the treatment indication and duration, as well as the potential adverse effects, were extensively discussed.The prescribed antihormonal agent given was recorded. To systematically evaluate the different clinical situations during the course of adjuvant endocrine therapy, we created the following subdivisions:(A) Patients who did not initiate therapyThis subgroup includes the patients to whom endocrine therapy was not recommended, and the patients who refused the recommended therapy and never began treatment.(B) Patients who initiated therapyThis subgroup includes the patients who completed the therapy after a 5-year course . Patients with extended therapy >5 years were also considered as having fully completed therapy. Further subgroups must also be considered as having completed therapy, although treatment was not administered during the targeted 5 years: discontinuation due to death, as well as discontinuation due to breast cancer recurrence  and serious medical reasons independent from breast cancer disease and therapy-related adverse effects. We distinguished the above-mentioned subgroups from non-adherent patients who discontinued the planned mode of treatment and refused to continue further endocrine therapy.Furthermore, we recorded any change of endocrine agents and the indication for the change: sequential therapy (switching after 2–3 years of adjuvant tamoxifen therapy to an aromatase inhibitor), extended therapy beyond 5 years of adjuvant therapy or change due to adverse effects. Lastly, we recorded the location of treatment and follow-up of the patients .Information concerning type and length of the medication, as well as the reasons for discontinuation, was retrospectively obtained from the medical record. Patients who had no follow-up at our institution were contacted via telephone. Afterwards, contact was made with the treating physician to confirm the patients' statements.The study design and data collection methods were approved by the Institutional Review Board.P-value less than 0.05 was considered significant. Statistical analyses were performed with R Development Core Team Software .To identify factors associated with treatment refusal and non-adherence, we created two univariate logistic regression models. Each model included the independent variables: year of the initial diagnosis, patient's age, primary surgical therapy, tumour stage, receipt of previous chemotherapy and/or postoperative radiation, and location of follow-up (the latter only in the non-adherence model). Odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were reported; a The clinicopathologic, treatment and follow-up characteristics of the 325 patients in the study are summarised in (A) Patients who did not initiate endocrine therapy (n=38)P=0.019; study period: 1997–2000 vs 2001–2003: P=0.0070; no chemotherapy: P<0.0001;P=0.0546).In 10 cases, despite HR-positive carcinoma, a recommendation for endocrine therapy was not made. The reasons for this included a low-risk constellation  and/or advanced age with considerable comorbidity. Twenty-eight patients (8.9%) refused the recommended endocrine therapy after extensive counselling and never began this treatment. Univariate analysis revealed that older patients, those who were initially diagnosed at the beginning of the study period and those who did not receive adjuvant chemotherapy were more likely to refuse therapy (advanced age: (B) Patients who initiated endocrine therapy (n=287)The initial endocrine agents prescribed are listed in In 50 patients, there was a change of the antihormonal agent prescribed within the planned 5-year period. Of these, four patients changed their medication twice and one patient three times. Twenty-five of the 50 patients changed their medication due to adverse effects of the endocrine therapy (three changed the medication twice and one three times). Of these, 20 patients (80%) fully completed the therapy after drug change.The therapy course, reported in n=5; incurable malignancy other than breast cancer, n=3; irreversible coma following severe head trauma, n=1).Of the 287 patients who initiated endocrine therapy, 191 (66.6%) fully completed the targeted therapy . Eleven patients (3.8%) discontinued the therapy due to death, whereas 43 patients (15.0%) ceased therapy due to breast cancer recurrence. In nine cases (3.1%), the therapy was discontinued by the physician due to serious medical reasons independent from breast cancer disease and therapy-related adverse effects  chose independently to end the therapy. The main reasons for this given by the patients are listed in P=0.0088; Location of follow-up: Of the 289 patients who received adjuvant endocrine therapy, 215 (74.4%) were treated in an oncology unit; of these, 194 patients in the internal oncology unit (67.1%) and 21 (7.3%) in an external oncologic unit. Seventy-two patients (24.9) had further follow-up and treatment through a general practitioner. Two patients (0.7%) were lost to follow-up, and the location of the oncologic care was unknown.All studies exclusively analysed selected study cohorts.Some studies used insurance claims and public health data  and those whose therapy had to be stopped due to breast cancer recurrence or other serious medical reasons  must not be analysed in one collective study group.A realisation of this goal requires a careful and detailed clinical follow-up of the study group and a clear discrimination between the terms and reasons behind ‘discontinuation’ and ‘non-adherence’. The currently available studies from clinical practise settings have not been able to accomplish this. In most of these studies, patients who discontinued therapy, regardless of the reason for cessation, were categorised in a single group, and the terms ‘discontinuation’ and ‘non-adherence’ were used synonymously   (Several studies performed in a clinical practise setting demonstrated that adherence to endocrine therapy was influenced by certain factors such as age, adverse effects, patient beliefs about the risks and benefits of tamoxifen use, history of medication and good patient–doctor relationship . Some ofAs most of the patients of our study were treated at the oncology unit of our department, the favourable non-adherence rates are probably associated with the fact that all practitioners received a targeted education in the techniques of patient-centred communication through the departmental division of psychosomatic medicine. These communication skills were applied in the follow-up of oncologic patients, in particular regarding the dialogue concerning possible therapy-related side effects and the importance of compliance and adherence to therapy . In our The limitations of our retrospective study, however, must be considered. First, our study relies on information obtained by patients' self-report of adherence. It is possible that in some cases, the patients who reported continuing to take medication had indeed stopped taking it and just gave a socially acceptable answer. Furthermore, we defined non-adherence as an intentional behaviour. Our data show that, when compared with other studies, low non-adherence rates can be realistically achieved. In the future, multifactorial approaches should be further analysed and refined with the goal of further improving compliance and adherence, and by doing so, the outcome of breast cancer patients .


Viruses can fall prey to their defective interfering (DI) particles. When viruses are cultured by serial passage on susceptible host cells, the presence of virus-like DI particles can cause virus populations to rise and fall, reflecting predator-prey interactions between DI and virus particles. The levels of virus and DI particles in each population passage can be determined experimentally by plaque and yield-reduction assays, respectively.To better understand DI and virus particle interactions we measured vesicular stomatitis virus and DI particle production during serial-passage culture on BHK cells. When the multiplicity of infection  was fixed, virus yields followed a pattern of progressive decline, with higher MOI driving earlier and faster drops in virus level. These patterns of virus decline were consistent with predictions from a mathematical model based on single-passage behavior of cells co-infected with virus and DI particles. By contrast, the production of virus during fixed-volume passages exhibited irregular fluctuations that could not be described by either the steady-state or regular oscillatory dynamics of the model. However, these irregularities were, to a significant degree, reproduced when measured host-cell levels were incorporated into the model, revealing a high sensitivity of virus and DI particle populations to fluctuations in available cell resources.This study shows how the development of mathematical models, when guided by quantitative experiments, can provide new insight into the dynamic behavior of virus populations. Defective interfering (DI) particles are virus-like byproducts of infections that are unable to infect cells on their own because they carry mutations, typically large deletions, in essential viral genes. However, when DI particles and standard virus particles co-infect the same cell the DI particles compete for resources that ultimately enable them to reproduce at the expense of virus particles. DI particles were discovered more than 60 years ago ,2, and tThe unique biological properties of DI particles make them interesting and important for several reasons. First, their ability to interfere with standard virus infections highlights their potential use as therapeutic agents . MoreoveA characteristic feature of DI particles is their emergence and outgrowth during high multiplicities of infection (MOI), where numerous copies of the standard virus infect each host cell. Under such conditions "predator" DI particles can productively interact with "prey" cells infected with standard virus. Ecological models of predator-prey behavior have been proposed to describe the dynamics of virus and DI particle populations -26, wher2 in Eagle's Minimum Essential media  with 5% GlutamaxTM (Gibco) with 10% fetal bovine serum ; the media was switched 2% FBS for all virus infections. All studies employed VSV-N1, a molecularly well-defined virus strain based on the San Juan isolate of the Indiana serotype of VSV [4 PFU/ml) to inoculate 8 × 106 cells in a T-75 flask , incubating 24 h at 37°C, passing through a 0.22 micron filter , and storing at -80°C. The stock titer was 1 × 109 PFU per ml, and the absence of detectable DI particle activity was confirmed by the yield-reduction assay.The maintenance and analysis of host cell and virus cultures followed established methods . Brieflye of VSV , generouThe interference of VSV infection by DI particles was quantified by the yield-reduction assay ,30. Prio6 cells per well in a six-well plate. For fixed-MOI cultures the virus level from each passage was determined by plaque assay and diluted using cell culture media with 2% FBS to set the MOI for the next passage. To infect, 100 microliters of the diluted virus solution was added to the cell monolayer. The dish was then swirled so that the virus solution would fully coat the cell monolayer. After incubation at 37°C for 1 h, the virus solution was removed, and 2 ml of cell culture media with 2% FBS was pipetted into the well. The wells were then incubated at 37°C for 23 h. After this incubation period the solution was mixed by pipetting, aliquoted, and stored at -90°C until diluted for titering or use in a subsequent infection. For fixed-volume passages, cell culture media containing 2% FBS was added to the well immediately upon removal of the media containing 10% FBS. Each passage infection was initiated by transferring 20 microliters of the mixed virus solution from the previous passage. The infection was then incubated at 37°C for 24 hours and harvested, as described above.Prior to each virus passage, fresh cells from parallel-prepared cultures were counted. Typical passage infections used 1.6 × 10As detailed in the Results section, difference equations linking inputs and outputs of infection were employed to estimate overall yields of virus and DI particles. Parameter values (p) were estimated by least squares:with the summation spanning the available data points. The corresponding confidence intervals were constructed by parametric bootstrapping percentile method using multiple resamples from the fitted model as detailed elsewhere .Over the course of 13 fixed-volume passages levels of virus production from cell monolayers dropped more than 10-fold, and passage-to-passage titers of virus fluctuated up to 100-fold Fig. , produciTo better understand how the accumulation of DI particles across fixed-MOI passages impacts virus yields we quantified the activity of interfering particles in different passage populations using the yield-reduction assay Fig. . At highz is the number of IUs per IE. The model equations are:To quantitatively assess the effects of virus-DI particle interactions during serial-passage culture we applied a mathematical model that we recently developed to quantitatively account for the input-output behavior of single passages . Brieflyth passage (VN+1) depends on virus amplification in cells initially infected with either virus alone (CV) or cells co-infected with both virus and IEs (CB). The output concentration of IEs from the Nth passage (IEN+1) depends on their average production from co-infected cells (CB) and their generation from IE-free infected cells (CV). To determine how the initial number of cells (CN) distribute among those that contain virus alone (CV) or virus and IEs (CB) we assume that particles encounter and infect cells following a Poisson distribution, as shown in equations 3 and 4. The model has nine parameters, presented in Table z, three parameters describe the productivity of virus, and five parameters describe the productivity of IEs. In the absence of DI particles a virus-infected cell produces an average of b virus particles. This productivity level is then modified by parameters p and q which describe how the virus productivity changes depending on the dose of interference. The negative sign on p reflects the reduction in virus yield from DI particle co-infection, and the positive value of q accounts for a partial recovery of virus yield for large co-infecting doses of DI particles. The IEs are either generated de novo from virus-infected cells (CV), described by the parameter g, or amplified from co-infected cells (CB), described by the parameter d. The effects of IE co-infection on IE yield is then modified by the parameters r, s, and w. At low levels IEs enhance their own production, reflected by the positive value of r. At higher IE, where N·z/CN)   × 106 cells per well, respectively.The impact of host-cell availability for infection was tested by simulating passages using either fixed or fluctuating cell numbers. Fluctuating conditions were simulated by using cell numbers that were experimentally measured during the fixed-volume passage, at the time of infection Fig. . Both vi4 IE per ml had no effect on the productivity of virus or IE at steady state. However, transients in virus and IE productivity differed in duration, requiring up to 9 passages to achieve steady-state levels.The model was used to simulate the effects of different initial IE levels on the behavior of virus and IE levels across fixed-volume passages, as shown in Fig. q = 0, rather than q = 0.47 , as shown in Fig. infected cell. Other mechanisms may also play a role in the mismatch between model and data at low MOI. For example, DI particle populations may evolve to more potently inhibit amplification of virus in cells co-infected with both virus and DI particles, or de novo generated DI particles may emerge and displace existing DI particles [By maintaining a fixed multiplicity of infection (MOI) across serial virus passages we observed a regular pattern of population behavior: average yields of virus declined more rapidly for passages performed at higher MOIs Fig. . The eararticles ,33,34. FN+1V) and cell level(NC) shown in Eq. 1. A testable prediction of the model is that by controlling experiments to reduce fluctuations in cell levels one should observe more regular behavior of virus levels  during constant-volume passages, as reflected in Fig. What causes the chaotic population dynamics that is a hallmark of fixed-volume serial virus cultures? Mathematical models of passage behavior have previously suggested how chaotic behavior can arise during fixed-volume passages , but thede novo generation of DI particles produced interference activity that was rapidly amplified in co-infections with wild-type virus. It appears that once DI particles are available they will tend to dominate subsequent co-infections. While new or different DI particles could, in principle, be generated by de novo processes, the low productivity of such processes prevents de novo DI particles from being competitive with existing DI particles. This dominance of initial DI particles has been found experimentally by observing how parallel cultures initiated from a common stock preserve DI particles present in the stock [To run our model one must provide an initial condition that specifies not only the concentration of virus particles, but also the concentration of DI particles in the initial passage. In the laboratory, the concentration of DI particles in the initial passage will depend on the history of the stock solution. For example, if the stock is amplified from an isolated plaque at low MOI or serially passaged at high MOI it may contain negligible or high levels of DI particles, respectively. In our model this input was found to have no effect on the steady-state dynamics of the virus and DI particle populations Fig. /4h. Evenhe stock .de novo rate of DI particle generation. This rate was estimated for VSV at 0.001 IE/cell. However, when a cell is co-infected with viable virus and DI particles, then many DI particles can be made, reflecting their ability to divert resources from virus to DI particle production.Some cells can make nearly a million-fold more DI particles than other cells, depending on how they are infected. When a cell is infected with viable virus, in the absence of any DI particles, few if any DI particles will be made, reflecting the small de novo rate of DI particle generation is (0.001 IE/cell × 200 IU/IE × 1 DI particle/IU) or 1 DI particle from every five infected cells. Thus, a 500,000-fold difference in DI particle yields separates the most productive from the least productive cells. A more expanded view of productivity, which includes the production of both DI and viable particles, may be concisely expressed in terms of a particle-to-PFU ratio. Such a ratio can be experimentally determined by estimating the number of virus-sized particles in a sample by electron microscopy and dividing by the number of viable virus particles measured by the plaque assay. If we assume that only DI and viable particles are produced by a co-infected cell, then we estimate a particle-to-PFU ratio of 38  based on maximal production of DI and viable particles. This estimate is consistent with the observed range for VSV from 10 to 50 particle-to-PFU [Experiments suggest that more than 500 IE per cell can be produced under optimal conditions for DI particle production . If we ae-to-PFU .q to zero, so cells co-infected with virus and a high level of DI particles would produce only DI particles. Others have found that individual cells isolated from a co-infected cell population produced no detectable virus, suggesting a curing effect, but other cells drawn from the same population did produce virus [Although spontaneous curing of virus infections by DI particles has not been experimentally observed, the possibility has been explored in previous models ,22. Our ce virus . The absce virus ,40, as pce virus . In an iWe are seeking to develop experimental measures and theoretical methods to better understand how viruses grow and how their infections spread. Our main contribution here has been to better link quantitative experiments of virus and DI particle populations with quantitative models. This approach will be enhanced in the future by incorporating additional measures, beyond plaque counting, including electron microscopy (particle counting), quantitative PCR, and next-generation sequencing. Ultimately, our approach aims to provide a predictive capability that will enable the long-term control of virus infections.Serial-passage cultures of virus at fixed multiplicity of infection (MOI) produced regular patterns of attenuated virus production across passages. These patterns of steeper declines in virus production at higher MOI could be accounted for by a mathematical model that incorporated interactions among virus particles, defective interfering (DI) particles, and cells to predict yields of virus and DI particle production from co-infected cells. Further, analysis of fluctuating virus populations provided evidence for a high sensitivity of virus and DI particle population dynamics to small changes in the levels of cellular resources.The authors declare that they have no competing interests.KT and JY conceived of the study, and KT performed all experiments. KT and JY analyzed the data and wrote the manuscript.


Resistance to chemotherapy is a major obstacle to the effective treatment of many tumor types. Although many anticancer therapies can alter tumor growth, in most cases the effect is not long lasting. Consequently, there is a significant need for new agents with low susceptibility to common drug resistance mechanisms in order to improve response rates and potentially extend survival. Approximately 30% of the women diagnosed with early-stage disease in turn progress to metastatic breast cancer, for which therapeutic options are limited. Current recommendations for first-line chemotherapy include anthracycline-based regimens and taxanes (paclitaxel and docetaxel). They typically give response rates of 30 to 70% but the responses are often not durable, with a time to progression of 6 to 10 months. Patients with progression or resistance may be administered capecitabine, gemcitabine, vinorelbine, albumin-bound paclitaxel, or ixabepilone, while other drugs are being evaluated. Response rates in this setting tend to be low (20 to 30%); the median duration of responses is <6 months and the results do not always translate into improved long-term outcomes. The present article reviews treatment options in taxane-resistant metastatic breast cancer and the role of ixabepilone in this setting. The development of chemotherapy resistance continues to be the main problem in the treatment of cancer patients. Newer agents, whether chemotherapeutic or targeted, are constantly being developed. Although most anticancer therapies will alter tumor growth, in most cases the effect is not long lasting and failure of anthracyclines and taxanes impact the survival of breast cancer patients. Consequently, there is a significant need for new agents with low susceptibility to common drug resistance mechanisms in order to improve response rates and potentially extend survival.Approximately 30% of the women diagnosed with early-stage disease in turn progress to metastatic breast cancer (MBC), for which therapeutic options are limited . After tResistance to chemotherapy can occur prior to drug treatment (primary or innate resistance) or may develop over time following exposure (acquired resistance) . PatientOnce resistance to taxanes or anthracyclines occurs, few treatment options exist. Most breast cancer patients with resistant or refractory disease are treated with capecitabine as a single agent or in combination. Approximately 75% of patients treated with capecitabine do not respond, and many responders eventually become resistant -10. OtheRecent research has suggested potential novel therapeutic targets for drug-resistant MBC. Tumor stem cells have been identified in many malignancies, including breast cancer ,20. AccuThe aim of the present article is to review the current therapeutic alternatives to treat MBC resistant to taxanes.Chemotherapy resistance can arise through a number of different mechanisms, including alterations in drug pharmacokinetics and metabolism, modification of drug target expression or function , drug compartmentalization in cellular organelles, altered repair of drug-induced DNA damage, changes in apoptotic signaling pathways , and expression of proteins directly affecting cellular drug transport (efflux pumps) Table  24,25].,25.24,25Many transport-mediated drug resistance mechanisms involve the ABC membrane transporter family. The most well-characterized examples of these drug efflux transporters include the P-glycoprotein (P-gp) pump, multidrug-resistant protein-1 (MRP1), and breast cancer resistance protein. These energy-dependent proteins actively pump drugs such as chemotherapeutics out of the cells, thereby reducing their intracellular drug concentration and decreasing the cytotoxicity .Expression of pumps such as P-gp or MRP1 gives tumor cells the ability to evade the chemotherapy drugs, and their role has been evaluated in breast cancer.MDR1 gene. This ATP-dependent membrane transporter pumps a diverse array of chemotherapeutics across the cell membrane and out of the cells, including anthracyclines, taxanes, vinca alkaloids, epipodophyllotoxins, and antifolates. The normal physiologic role of P-gp is still unknown, but it may serve to protect normal tissues from toxic products and xenobiotics .-92.88-922, infused over 3 hours, every 21 days for up to 18 cycles due to progressive disease. The overall response rate (ORR) was the primary endpoint.Given its activity in taxane-resistant breast cancer models, ixabepilone was clinically evaluated in patients with MBC resistant to taxane therapy. An international, multicenter phase II trial evaluated single-agent ixabepilone in patients with MBC who were previously treated with an anthracycline-based regimen and were resistant to a taxane . PatientMost patients in this study had been treated with at least two prior chemotherapy regimens. All of the patients had received at least one prior taxane-containing regimen (31% had at least two regimens), and 98% of patients had a taxane-containing regimen as their most recent therapy in the metastatic setting. This population was highly refractory because 73% of the patients had progressed within 1 month of their last administered taxane dose.Of the 49 patients eligible for efficacy analysis, there were six responses (ORR 12%) with a median duration of response of 10.4 months. All of the responders had extensive baseline disease and had failed multiple therapies. An additional 20 patients (41%) had stable disease as their best response. The median time to progression was 2.2 months , and the median survival was 7.9 months . Responses seen with ixabepilone in patients with taxane-resistant MBC confirm its clinical activity in this patient population and support its differential sensitivity to the mechanisms of resistance.2 was administered as a 3-hour intravenous infusion on day 1 of a 21-day cycle. The primary study end-point was the ORR.The largest phase II trial evaluated single-agent ixabepilone in patients with heavily pretreated or locally advanced disease or MBC resistant to the three standard chemotherapeutics in this setting; that is, anthracyclines, taxanes, and capecitabine . Resista77% of patients, and more than 40% had at least three target lesions. The majority of the patients (88%) had completed at least two prior chemotherapy regimens for MBC, and 48% had at least three therapy lines; 15% and 30% of patients had at least one line of anthracycline therapy and taxane therapy, respectively. All but two treated patients had taxane-resistant disease, while 38% had anthracycline-resistant tumors. Many had failed prior chemotherapy for MBC including vinorelbine (25%), gemcitabine (13%), and trastuzumab for HER2-positive disease (9%).The patients in this study had significant and widespread baseline disease: visceral disease in the liver and/ or lung was present in Of the 126 patients enrolled, 113 were evaluable for a response. As assessed independently, the ORR was 11.5%  with another 50% of the patients achieving stable disease as their best response. Tumor responses were durable, with a median duration of 5.7 months ; eight of the 13 responders remained progression free for ≥6 months. The median progression-free survival (PFS) was 3.1 months , and the median overall survival was 8.6 months . Ixabepilone monotherapy was therefore active in patients with difficult-to-treat, advanced, highly refractory breast cancer who had failed to respond to prior chemotherapy. One should note that nine of the 12 responders to ixabepilone had not responded to prior multiple lines of chemotherapy in the metastatic setting, including combination regimens.2, infused over 3 hours on day 1, in addition to capecitabine 2,000 mg/m2 on days 1 to 14, both given every 21 days. Patients were ineligible if they had received more than three prior chemotherapy regimens for metastatic disease.Given the single-agent activity of ixabepilone in women previously treated with anthracyclines, taxanes, and capecitabine, and the need for more effective second-line MBC regimens, the combination of ixabepilone and capecitabine was evaluated in phase II and phase III trials. In the phase II study, patients previously treated with anthracyclines and taxanes were treated with ixabepilone in addition to capecitabine . Sixty-tFifty patients were evaluable for a response: 72% had baseline visceral metastases, and 42% received at least two prior chemotherapy regimens for metastatic disease. Fifteen responses occurred (30% ORR), and stable disease was achieved in 32% of patients. All of the responders had extensive metastatic disease at baseline. The median time to response was 6 weeks , with most responses occurring by the end of the second cycle. The median duration of response was 6.9 months . Four of the 15 responses occurred in patients with estrogen receptor-negative, progesterone receptor-negative, and HER2-negative (triple-negative) breast cancer, suggesting such a regimen may be effective for patients with this treatment-resistant subtype . These p2, administered as a 3-hour infusion on day 1 of a 21-day cycle, plus capecitabine 2,000 mg/m2 on days 1 to 14 of a 21-day cycle. Those patients on capecitabine alone were administered a dose of 2,500 mg/m2 on days 1 to 14 of a 21-day cycle. The primary endpoint was PFS. The patients enrolled in this study (n = 752) had widespread disease and were heavily pretreated with chemotherapy. Most patients (65%) had at least three metastatic disease sites, and nearly one-half had received at least two prior regimens for metastatic disease. The majority of patients (85%) had progressed on prior taxane therapy for MBC.These encouraging phase II results led to an international, randomized, open-label phase III trial that compared ixabepilone plus capecitabine with solely capecitabine administration in patients with locally advanced or MBC pretreated with or resistant to anthracyclines and taxanes . PatientP = 0.0003), in turn reflecting a 25% reduction in the estimated risk of disease progression  compared with capecitabine alone (14%); stable disease occurred in 41% and 46% of patients, respectively. The combination regimen demonstrated activity in triple-negative disease, confirming the activity observed in this subgroup in the phase II trial. Mature overall survival data are anticipated within several months. The most frequent grade 3/4 adverse events in the ixabepilone plus capecitabine group were peripheral sensory neuropathy (with a median onset of four cycles), hand-foot syndrome, fatigue, myalgia, asthenia, and diarrhea; while the most frequent grade 3/4 adverse events in the capecitabine group were hand-foot syndrome and diarrhea, but with incidences similar to those for the combination arm. The incidence of adverse events commonly associated with capecitabine, such as hand-foot syndrome, were not exacerbated by the addition of ixabepilone.The trial results demonstrated that PFS significantly improved for patients treated with ixabepilone plus capecitabine compared with capecitabine alone  has been studied in a phase II study of weekly albumin-bound paclitaxel for patients with MBC heavily pretreated with taxanes. Response rates were 14% and 16% for the 100 mg/m2 and 125 mg/m2 cohorts, respectively; an additional 12% and 21% of patients, respectively, had stable disease with an acceptable toxicity profile [While ixabepilone is being evaluated in combination with other drugs, new drugs are currently being tested and have the potential to become standard treatments in this MBC setting. Albumin bound paclitaxel  polymerase inhibitors are one group of drugs with great potential in resistant breast cancer, especially triple-negative and BRCA-deficient breast cancer. A phase II study of olaparib in confirmed BRCA1/ BRCA2 carriers with advanced refractory breast cancer showed an ORR of 38% . Other pDrug resistance (primary or acquired) is a leading cause of treatment failure in patients with cancer, especially MBC. Patients with advanced or MBC commonly develop disease resistance to chemotherapy and even biologic therapies such as trastuzumab, leaving few effective treatment options. The occurrence of MDR disease in many patients with advanced breast cancer due to the overexpression of βIII-tubulin isotype or drug transporters, such as P-gp, demands new approaches. Consequently, there is a significant need for novel agents that are effective in drug-resistant tumors with mechanisms of action that are different from the available chemotherapeutics.The epothilone B analog ixabepilone demonstrates significant antitumor activity against a variety of tumor cells with primary or acquired drug resistance, including MDR. Ixabepilone is less susceptible to the common mechanisms of drug resistance, particularly tubulin mutations, compared with taxanes and other traditional chemotherapy. Clinical trials demonstrate single-agent ixabepilone to be active in MBC patients with highly resistant or refractory disease who have a significant tumor burden. Antitumor activity was observed in those patients who have had extensive prior therapy with anthracyclines, taxanes, and/or capecitabine. Ixabepilone toxicity was manageable and comparable with other commonly used chemotherapeutics for MBC. In combination regimens, ixabepilone plus capecitabine resulted in greater activity compared with capecitabine alone in a taxane-resistant population, without significantly increasing toxicity. Ixabepilone has been approved by the US Food and Drug Administration for use in combination with capecitabine for the treatment of locally advanced breast cancer or MBC after the failure of an anthracycline and a taxane, and as monotherapy after the failure of an anthracycline, a taxane, and capecitabine. A previous publication suggests that the cost-effectiveness ratio could be higher for addition of ixabepilone to capecitabine treatment .The potential of ixabepilone in patients with early-stage breast cancer is currently under evaluation. Given the clinical impact of drug resistance in breast cancer and other malignancies, new agents are clearly needed with differential sensitivity to the various mechanisms of tumor resistance compared with the standard chemotherapy drugs. Increased application of pharmacogenomics may also allow for the identification of patients with, or at increased risk for, drug resistance as well as those who are most likely to benefit from the treatment.ABC: ATP-binding cassette; CI: confidence interval; HER2: human epidermal growth factor receptor-2; MBC: metastatic breast cancer; MDR: multidrug resistance; MRP1: multidrug-resistant protein-1; ORR: overall response rate; PCR: polymerase chain reaction; PFS: progression-free survival; P-gp: P-glycoprotein; RT: reverse transcription.The author receives research grants from Bristol Myers.Published: 22 October 2010


Magnetic resonance-guided focused ultrasound surgery (MRgFUS) is a non-invasive treatment approach for symptomatic uterine fibroids. One imaging characteristic considered in selecting patients who may benefit from MRgFUS of their uterine fibroids is the signal intensity of the fibroid compared with surrounding myometrium on T2-weighted MR images. Previous reports suggest that hyper-intense fibroids are less amenable to MRgFUS compared with iso- or hypo-intense fibroids. In this case study, we utilized contrast-enhanced dynamic MR imaging to further characterize the vascularity of a hyper-intense fibroid. Based on the results of dynamic T1-weighted contrast-enhanced images, we assumed that the hyper-intense appearance resulted from high fluid content rather than high vascularity and predicted that the fibroid would respond to MRgFUS. The patient underwent the MRgFUS without complication and reported significant decrease in fibroid symptoms at 3 and 12 months post-treatment. This case suggests that pre-treatment dynamic contrast-enhanced imaging used in conjunction with T2-weighted imaging may improve the criteria for selecting uterine fibroids amenable to treatment with MRgFUS, potentially leading to improved patient outcomes. Uterine fibroids are the most common tumors of the female reproductive tract. Fibroids have been identified clinically in at least 25% of women , and patSeveral options, each with varying degrees of invasiveness, are available for treatment of symptomatic uterine fibroids. These include among others, hysterectomy, myomectomy , uterine artery embolization, MR-guided Focused Ultrasound (MRgFUS), and hormonal therapy , which aMRgFUS is a non-invasive treatment for uterine fibroids. It utilizes precisely focused ultrasound waves to generate and maintain high temperatures within the targeted fibroid, resulting in protein denaturation and coagulative necrosis . MRgFUS While clinical studies demonstrate that MRgFUS is a safe and effective treatment for symptomatic uterine fibroids –8, not aOne imaging characteristic considered in the patient selection process is the fibroid's capacity to absorb heat, as the effects of MRgFUS result from the thermal ablation of the fibroid tissue. This is often assessed based on the signal intensity of the fibroid compared with the surrounding myometrium on T2-weighted MR images (T2WIs). Previous reports suggest that the average nonperfused volume (NPV), as measured by posttreatment contrast-enhanced image, obtained with MRgFUS in the treatment of hyper-intense fibroids, is lower than that achieved in iso- or hypointense fibroids , 11. The following case report demonstrates successful treatment of a fibroid that is hyper-intense on T2WIs and suggests that contrast-enhanced dynamic MR imaging may be used to select a subpopulation of uterine fibroid patients suitable for MRgFUS despite hyper-intense results on T2WIs.A 47-year old premenopausal Asian female, with BMI of 20, was referred to our clinic by her gynecologist due to severe menorrhagia. Her Symptom Severity Score  was 56 To evaluate the fibroid's viability more fully, and as part of our screening routine for MRgFUS, T1-weighted MR images (T1WIs) with contrast were acquired. The contrast agent used was Optimark . Dynamic T1-weighted gradient echo sagittal images were acquired every 15 seconds from the moment of injection until 180 seconds after the injection  . T1-weigAfter consulting with the patient about the potential benefits and risks involved in treating her specific fibroid, she chose to undergo MRgFUS, knowing that results might be less than satisfactory due to the hyper-intense appearance of her fibroid. Treatment was performed using the ExAblate 2000 system  and SIGNA HDx MRI . T2WIs were acquired for treatment planning. The procedure required 128 focal ablations (sonications) with average energy of 3300 Joules. Treatment duration was 3.5 hours from first to last sonication. Average temperature achieved was 75°C. Contrast-enhanced T1WIs were acquired posttreatment, showing that 335 cc (60%) of the fibroid was nonenhancing .The treatment was concluded without complications. During her followup phone call the next day, she reported returning to work and feeling well with no adverse events. The patient provided a similar report in a phone call 7 days posttreatment. At the 3-month followup, the patient's symptoms significantly improved, and her SSS score was reduced to 28 points. At that time, the patient returned for MR imaging, which showed 38% fibroid shrinkage. At 1-year followup, which included a clinical visit to evaluate patient's symptoms, her SSS was reduced to 13 points, which is considered as nonsymptomatic.In MRgFUS for symptomatic uterine fibroid, the non-perfused volume (NPV) of uterine fibroids has been found to correlate with treatment-related symptom relief, and to be inversely related with the need for repeated treatment  . The sucBased on previous experience, MRgFUS treatment of hyperintense fibroids usually results in relatively poor NPVs, and subsequent treatment failures . The hypThis case report shows that hyper-intense fibroids can be successfully treated with MRgFUS, however, the fibroids should be subcategorized based on their potential for successful treatment. T2WIs contribute to important patient-selection information regarding the location and structure of the fibroid(s) and coexisting pathologies. T1WIs with contrast examine the viability of the fibroid and, when performed dynamically, provide additional information on the rate of fibroid enhancement. Delayed enhancement may indicate that the fibroid has low vascularity and will absorb sufficient ultrasound energy to result in successful MRgFUS, even if the fibroid appears hyper-intense on the T2WIs.We suggest that dynamic contrast enhancement imaging, in addition to fibroid intensity in T2WI, may provide important information whether or not a fibroid is suitable for MRgFUS. Although in this case report, other factors could have contributed to the treatment success, we assume that the dynamic contrast imaging during screening helped in patient selection. Additional studies are needed to verify this theory and establish a correlation between the pretreatment dynamic contrast enhancement imaging characteristics of a given fibroid and patient outcomes following MRgFUS.


MicroRNAs (miRNAs) are short non-coding RNAs playing an important role in post-transcriptional regulation of gene expression. We have previously shown that hepatic transcript profiles are different between males and females; that some of these differences are under the regulation of growth hormone (GH); and that mild starvation diminishes some of the differences. In this study, we tested if hepatic miRNAs are regulated in a similar manner.Using microarrays, miRNA screening was performed to identify sex-dependent miRNAs in rat liver. Out of 324 unique probes on the array, 254 were expressed in the liver and eight (3% of 254) of those were found to be different between the sexes. Among the eight putative sex-different miRNAs, only one female-predominant miRNA (miR-29b) was confirmed using quantitative real-time PCR. Furthermore, 1 week of continuous GH-treatment in male rats reduced the levels of miR-451 and miR-29b, whereas mild starvation (12 hours) raised the levels of miR-451, miR-122a and miR-29b in both sexes. The biggest effects were obtained on miR-29b with GH-treatment.We conclude that hepatic miRNA levels depend on the hormonal and nutritional status of the animal and show that miR-29b is a female-predominant and GH-regulated miRNA in rat liver. In the latest release of miRBase , a sequence database of miRNA hosted by the Sanger Institute . If the Some miRNAs are ubiquitous, such as let-7b and miR-22 , while oThe liver is a major metabolic organ, handling the metabolism of exogenous and endogenous compounds. We and others have shown that male and female rat livers differ regarding gene expression and metabolism -10. We hInitially, we screened for miRNAs with a sex-different expression using microarray analysis. We generated hepatic miRNA profiles from normal male and female rats. Out of 324 unique probes on the array, 254 (78% of 324) were expressed in the liver and eight (3% of 254) of those were found to be different between the sexes Table . Among tTo validate our findings, miRNAs from Table GH is one of the hormones that has been shown to regulate many male or female-predominant gene products in rat liver  and, dueWe have previously compared gene expression levels from rat livers during mild starvation (12 hours of food deprivation) and the postabsorptive state (4 hours of food deprivation) . The notIn this study, a miRNA screening was performed to identify sex-dependent miRNAs in rat liver. Among eight putative sex-different miRNAs, at least one female-predominant miRNA (miR-29b) could be confirmed using qRT-PCR. In contrast, the candidates for male-predominant miRNA could not be confirmed. In order not to miss any putative sex-dependent miRNA, a relatively high false discovery rate (q < 10%) and low cut-off (1.5) regarding differences between male and female expression levels were used. Moreover, the chemistry for detection by microarray and qRT-PCR are different. These might explain the inconsistent results between microarray and qRT-PCR, and stresses the importance of validating results obtained in this type of screening experiments.miR-122a might be another female-predominant miRNA in rat liver, since significantly higher  levels were observed in females than in males in the postabsorptive state. We observed smaller individual differences in miR-122a levels in these animals compared to animals with free access to food, which could be related to smaller individual differences in their metabolic states. This together with reports on multiple transcript variants of miR-122 [The female-predominant expression of miR-29b and miR-122a observed in this study might be of importance for metabolic control, since these miRNAs were shown to be increased during mild starvation, a condition when the liver switches from anabolic to catabolic pathways. In an as yet unpublished study, we have observed that these mildly starved rats have lower hepatic glycogen content and higher rates of lipid turnover, as compared to the postabsorptive rats. Responses to mild starvation were also observed at the level of hepatic gene expression (unpublished results). However, since most transcript levels were independent of food deprivation, we do not believe that the data presented in figure de novo lipid synthesis. This is in line with the observation that GH-treated male rats have higher hepatic triglyceride content [Among the miRNAs subjected to qRT-PCR, miR-29b was shown to be female-predominant and down-regulated in males given GH treatment. Continuous infusion of GH to male rats has previously been shown to mimic the female-specific endogenous secretion of GH and to partly "feminize" hepatic gene expression in rodents -16. Obvi content .in silico prediction analysis and in vivo data on miRNA targets is large [in vivo studies should always be performed to validate any predicted functions and actions of miRNA.To address the question of a putative biological significance of sex-different hepatic miRNAs, we searched for miRNA targets using the online databases PicTar  and Targis large . This diis large . Neverthin silico and in vivo studies, He et al identified a potential miR-29 target, insulin-induced gene 1  [Combining in CL-6) . INSIG1 in CL-6) . We havein CL-6) , INSIG1 We have previously shown that hepatic transcript profiles are different between males and females; that some of these differences are under the regulation of GH; and that mild starvation diminishes some of the differences. In this study, we show that hepatic miRNAs are regulated in a similar manner.Ten weeks old male and female Sprague-Dawley rats  were maintained under standardized conditions. Five male rats were treated with bovine GH (bGH), a kind gift from Pharmacia and Upjohn AB, by continuous infusion from osmotic minipumps  at a dose of 5 μg/h. After one week of treatment, the rats were sacrificed and tissues removed and frozen in liquid nitrogen. When the effect of mild starvation was investigated, another group of age-matched male and female rats were used. Food was removed early in the morning (7 a.m.) or late in the evening (11 p.m.), so that the animals were without food 4 hours (postabsorptive) or 12 hours (mild starvation) before they were sacrificed (around noon). Animals were anesthetized, sacrificed, tissues removed and immediately frozen in liquid nitrogen and stored at -80°C until further analysis. All animal experiments were approved by the institutional animal care and use committee.miRNA-enriched RNA was extracted from liver using miRNeasy Mini kit (Qiagen), according to the manufacturer's protocol. In short, 25 mg of frozen liver was homogenized and extracted using phenol:chloroform. The resulting aqueous phase was mixed with 80% ethanol to extract RNA, followed by precipitation using 100% ethanol and high salt buffers. One of these buffers removes large RNAs (> 200 nucleotides). After a wash using 80% ethanol, miRNA-enriched RNAs were eluted using RNase-free water and quantified by a NanoDrop ND-1000 Spectrophotometer Locked Nucleic Acid) microRNA Arrays , according to the manufacturer's protocol. miRNA was considered to be expressed if it was detected in more than 75% of the individual array experiments with a reliable signal . The data were normalized by the algorithm LOWESS using the program R and the significance of any sex-different expression was calculated using the program Significance Analysis of Microarray (SAM). miRNAs identified with a false discovery rate  lower than 10% and a male to female ratio higher than 1.5 were considered to be significantly sex-different. A p-value was calculated comparing the overall ratio to a value of 1.0, using student's t-test.Four individual arrays were run for each sex. 3 μg of miRNA-enriched hepatic RNA derived from four males and four females were labeled and hybridized against each other as individual pairs onto the miRCURY™LNA  were extracted and analyzed twice. The graphs show the average of at least eight data points for each group and the error bars represent their S.E.Selected miRNAs , and 5S rRNA as reference gene, were further examined using the miRCURY™LNA Real-time PCR kit , according to the manufacturer's protocol. Briefly, 100 ng miRNA-enriched RNA was reverse-transcribed into cDNA using miRNA-specific primers for miRNA detection or random hexamer for the reference gene. The reverse transcription primer carries an additional sequence complementary to the 3'-end of the PCR primer. Together with a miRNA-specific LNA primer, the synthesized cDNA was amplified and detected by SYBRLC took part in the design of the study, carried out animal experiments, RNA isolation, cDNA synthesis and real-time PCR analyses, immunoblotting and drafting of the manuscript. CG took part in animal experiments, carried out RNA isolation, cDNA synthesis and microarray analyses. GN participated in design and coordination of the study, took part in animal experiments and helped to draft the manuscript. PTE participated in design and coordination of the study, took part in animal experiments and drafted the manuscript. All authors read and approved the final manuscript.


Identification of disease susceptible genes requires access to DNA from numerous well-characterised subjects. Archived residual dried blood spot samples from national newborn screening programs may provide DNA from entire populations and medical registries the corresponding clinical information. The amount of DNA available in these samples is however rarely sufficient for reliable genome-wide scans, and whole-genome amplification may thus be necessary. This study assess the quality of DNA obtained from different amplification protocols by evaluating fidelity and robustness of the genotyping of 610,000 single nucleotide polymorphisms, using the Illumina Infinium HD Human610-Quad BeadChip. Whole-genome amplified DNA from 24 neonatal dried blood spot samples stored between 15 to 25 years was tested, and high-quality genomic DNA from 8 of the same individuals was used as reference.Using 3.2 mm disks from dried blood spot samples the optimal DNA-extraction and amplification protocol resulted in call-rates between 99.15% – 99.73% , and conflicts with reference DNA in only three per 10,000 genotype calls.Whole-genome amplified DNA from archived neonatal dried blood spot samples can be used for reliable genome-wide scans and is a cost-efficient alternative to collecting new samples. Studies of genetic influence in complex disorders usually require extensive genome explorations of large cohorts. A major bottleneck, however, is access to DNA from well-characterised patients and healthy controls. This may be circumvented by use of archived residual blood samples from newborn screening programs, which in several countries engage the entire population. The blood is usually collected by heel-prick and applied on special filter paper, a proven robust and convenient medium for transport and storage . StorageThe Danish Neonatal Screening Biobank (DNSB) contains nearly 2 million DBSS from virtually every Dane born after 1982. It has recently been updated to meet the new general guidelines for the establishment and operation of biobanks . The majIn this study we investigate if a proper combination of DNA-extraction and WGA procedure can produce wgaDNA samples suitable for 610,000 SNP genome-wide scan using the Illumina Infinium HD Human610-Quad BeadChip. Neonatal DBSS stored for 15 to 25 years in the DNSB are employed, and as reference is used high-quality gDNA samples recently obtained from the same individuals. Two different WGA methods are tested. The multi-displacement amplification (MDA) method (the REPLI-g kit) that produces relatively long wgaDNA fragments > 10 kb , and theThe investigation comprised 24 subjects born between 1982 and 1992, who all had their residual neonatal DBSS stored at -24°C in the DNSB. Four subjects were informed volunteers and 20 were from a genetic study on schizophrenia .® 16 Blood DNA Purification Kit (Promega). Neonatal DBSS from the eight participants were retrieved from the DNSB, and DNA was extracted from one or three DBSS disks, 3.2 mm in diameter, using Extract-N-Amp Blood PCR Kit (ENA)(extraction volume: 200 μL) (Sigma-Aldrich) or QIAamp DNA Blood Micro Kit (QIA)(extraction volume: 75 μL) (Qiagen). The DNA extracts were amplified using the REPLI-g kit (Qiagen), GenomePlex® Complete WGA Kit , or GenomePlex® Single Cell Whole Genome Amplification Kit . All procedures were performed according to the manufacturer's instructions. Prior to DNA-extraction, a subset of disks was extracted for proteins as described by Skogstrand et al. 2005 [® dsDNA Reagent .Reference gDNA was purified from venous blood samples from the four volunteers and from four subjects from the schizophrenia study using the Maxwell 16 automatic system and the Maxwellal. 2005 . Please The gDNA and wgaDNA samples were marked on the Illumina Infinium HD Human610-Quad BeadChip (Illumina) according to the manufacturer's instructions, with the exception that 240 ng of wgaDNA starting material was used instead of the prescribed 200 ng. The BeadChips were scanned using the BeadStation 500GX (Illumina) with a high-density upgrade and an AutoLoader (Illumina). The BeadStudio v.3 software (Illumina Corp.) was used for calculating call- and conflict-rates. In the first part of the study all calls were made using the reference Human610-Quadv1B cluster file from Illumina that is constructed from gDNA. In the second part of the study two cluster files, each constructed from 16 wgaDNA preparations made by the REPLI-g and GPlex4 kits (tailored cluster files specific for WGA method), were also used to analyse the respective samples. Conflict-rates were estimated comparing the wgaDNA samples to their respective reference gDNA samples. The percentage of conflicts introduced due to an allelic dropout (eg. AB to AA) was estimated by re-coding the Illumina data to variables allowing comparison using STATA v.9.0.The genotyping performance of the different wgaDNA preparations is seen in Additional file Combining REPLI-g WGA with QIA DNA-extraction was less successful and the results were highly variable. The genotyping performance using wgaDNA made by the two OmniPlex method kits, GPlex2 and GPlex4, was independent of the DNA-extraction method, with GPlex4 showing consistently higher call rates than GPlex2 . The reference gDNA call-rates were 99.8–99.9%. Almost all conflicts between results from the wgaDNA preparations and the reference gDNA were due to an allelic dropout (data not shown). Notably, extraction and amplification procedures that produced high call-rates displayed low conflict-rates with the reference gDNA and vice versa, which indicates that genome-wide scans on wgaDNA are reliable when the call-rates are high [Additional file Based on the results displayed in Additional file We demonstrate that wgaDNA, made from 3.2 mm disks of DBSS that have been stored at -24°C for more than 20 years is well suited for reliable genotyping of 610,000 SNPs, with call-rates comparable to those obtained using gDNA. The accuracy of genotype calls using wgaDNA from stored DBSS has been of some concern. The issue has been addressed several times, using both low and medium throughput genotyping platforms, and overall with good success ,12,14-18Because the investigation focuses ondifferent combinations of wgaDNA preparation, it suffers from the weakness that the number of samples in each group is limited. In addition, only samples from the DNSB were used.The combination of the ENA DNA-extraction with either the REPLI-g or the Gplex4 WGA kit were selected to see if the procedures were robust enough for GWA studies employing numerous samples. Both set-ups produced wgaDNA from 16 DBSS stored for 15 to 25 years that performed well with constant high call-rates. Corresponding reference gDNA samples were not available. Notably, when calling genotypes of wgaDNA preparations with the BeadStudio software, albeit clusters were nice and tight for some loci they did not fit well into the standard cluster positions. This is because the BeadStudio software calls the genotypes of a given locus by comparing the observed values with the expected values, defined by the Human610-Quadv1B Cluster file, which is based on gDNA samples ,23. In sEighteen WGA reactions, each producing ~5 μg of wgaDNA, can be made per ENA DNA extraction. As the Illumina Infinium HD Human610-Quad BeadChip uses 240 ng of wgaDNA, one WGA reaction is enough to run 20 chips. Thus one to three 3.2 mm disks from a DBSS are sufficient to make repeated GWS as well as fine-mapping genotyping, if required. We have briefly tested the performance of the two wgaDNA preparations on the Affymetrix platform and found that wgaDNA produced by the OmniPlex method was unsuitable, whereas wgaDNA produced by the MDA method gave results comparable to those obtained by the Illumina platform. In addition to being used for GWS, DBSS can also be used for multiplex protein measurements , quantitThe results demonstrate that residual DBSS from neonatal screening that have been stored for several years in biobanks can be used for GWS and hence for large genome-wide association studies. Using DBSS instead of collecting new samples may, in a cost-efficient way, reveal important correlations between genotypes, environment and human diseases. Both the OmniPlex and the MDA method performed excellently in combination with the ENA extraction, and we recommend to test which of the two WGA procedures is most suitable for a given task.MVH prepared the DNA samples, participated in the design of the study and did the major part of data analysis and drafting the manuscript. JG conducted the Illumina GWS and participated in the data analysis. AB, MN, BNP, TFO, PMB, CW, OM, MD and PT initiated the study and participated in its design and the interpretation of results. DMH initiated the study, participated in its design, the interpretation of results and drafting the manuscript. All authors were involved in a critical revising and approved the final manuscript.Performance of different wgaDNA preparations. 1 Year the DBSS was stored. 2 Kit used for DNA-extraction, QIA-QIAamp DNA Blood Micro Kit; ENA-Extract-N-Amp Blood PCR Kit. 3 Extraction of proteins from DBSS prior to extraction of DNA. 4 Number of 3.2 mm disks used. 5 DNA (ng) utilized per WGA reaction. 6 GWS genotypes call-rate (percent). 7 Rate of conflicts (percent) between genotype results on wgaDNA and reference gDNA. 8 wgaDNA (μg) produced per reaction.Click here for file


Women who have inherited mutations in the BRCA1 or BRCA2 genes have substantially elevated risks of breast and ovarian cancer. Mutation carriers have various options, including extensive and regular surveillance, chemoprevention and risk-reducing surgery. The aim of this review is to provide an up-to-date analysis and to subsequently summarise the available literature in relation to risk-reducing strategies, with a keen focus on prophylactic surgery.The literature review is facilitated by Medline and PubMed databases. The cross-referencing of the obtained articles was used to identify other relevant studies.Prophylactic surgery  has proved to be the most effective risk-reducing strategy. There are no randomised controlled trials able to demonstrate the potential benefits or harms of prophylactic surgery; therefore, the evidence has been derived from retrospective and short follow-up prospective studies, in addition to hypothetical mathematical models.Based on the current knowledge, it is reasonable to recommend prophylactic oophorectomy for BRCA1 or BRCA2 mutation carriers when childbearing is completed in order to reduce the risk of developing breast and ovarian cancer. In addition, women should be offered the options of rigorous breast surveillance, chemoprevention with anti-oestrogens--especially for carriers of BRCA2--or bilateral prophylactic mastectomy.The selection of the most appropriate risk-reducing strategy is not a straightforward task. The impact of risk-reducing strategies on cancer risk, survival, and overall quality of life are the key criteria considered for decision-making. Notably, various other factors should be taken into consideration when evaluating individual mutation carriers' individual circumstances, namely woman's age, morbidity, type of mutation, and individual preferences and expectations.Although prospective randomised controlled trials concerned with examining the various interventions in relation to the woman's age and type of mutation are needed, randomisation is extremely difficult and rather deemed unethical given the current available evidence from retrospective studies. Breast cancer remains the leading cause of death in women aged 40-55 years. SporadiHistologically, BRCA1-related breast cancers are predominantly of the basal subtype, with predominant lymphocytic infiltration, and are often more aggressive and associated with negative prognostic factors, as characterised by numerous mitoses, pleomorphic pattern, poor differentiation and higher proliferation rates -11, as wClinically, breast cancer is believed to begin at an earlier age in BRCA1 and BRCA2 gene mutation carriers compared with sporadic cases. The median age of breast cancer onset ranges from 40-50 years in BRCA1 and BRCA2 carriers compared with 60-70 years in sporadic cases,19. Furtet al., for example, recently conducted a meta-analysis of 11 previously published articles [Several studies have suggested no significant differences between BRCA-associated breast cancer and non-BRCA associated breast cancer in relapse-free, event-free, and overall survival -24. On tarticles . The anaet al. observes that short-term progression-free survival in BRCA1 carriers was worse than in non-BRCA carriers (HR = 1.54). Such observations were not witnessed in long-term progression survival. In addition, BRCA2 mutation was not observed to affect either short- or long-term survival rates, which may be attributed to the different carcinogenic pathways for BRCA1 and BRCA2 [Moreover, Lee nd BRCA2 .et al. suggest that peritoneal papillary serous carcinoma risk amongst BRCA2 carriers is lower than amongst BRCA1 carriers[Most ovarian cancers associated with germ line BRCA mutations are diagnosed at a younger age and are ultimately determined as high-grade and advanced-stage serous carcinomas. They ha carriers. Further carriers. Notably carriers, and adv carriers.The identification of BRCA1 and BRCA2 genes in 1994 and 1995 respectively,35 has hWomen who have an increased risk of breast and ovarian cancer are advised to consider risk-reducing strategies; however, such methods vary in their effectiveness. These strategies include surveillance , trans-vaginal ultrasound scanning and serum CA125), chemoprevention and prophylactic surgery ,39. Risk25, chemoThe aim of this review is to provide an up-to-date analysis and to subsequently summarise the available literature in relation to risk-reducing strategies, with a keen focus on prophylactic surgery.MEDLINE and PubMed were used to search for relative articles. Articles identified using the key words "breast cancer", "BRCA1", "BRCA2", "ovarian cancer", "risk reducing", "prophylactic", "mastectomy", "oophorectomy" and "life expectancy". Cross referencing was performed to identify other articles not identified at the initial search.The efficacy of surveillance in BRCA1/2 mutation carriers is difficult to determine. Importantly, it has been suggested that some BRCA1/2 carriers will not develop cancer during their lifetime, althougImportantly, annual ovarian surveillance--conducted by pelvic examination, trans-vaginal ultrasound scanning and serum CA125 measurement--in women at increased familial risk of ovarian cancer is found to be ineffective in detecting tumours at a sufficiently early stage, which substantially influences survival in BRCA1/2 mutation carriers-52. SeruIt is well recognised that oestrogen plays an essential role in mammary carcinogenesis by exerting a carcinogenic effect (through metabolites) on cellular DNA and thereby promoting the growth of ER positive breast cancer. TherefoProphylaxis with SERMs was estimated to prolong survival by 1.6 to 2.2 years in BRCA gene mutation carriers. The ablTargeting oestrogen synthesis is considered to be an alternative to SERMs as a chemo-preventative risk-reducing strategy in high-risk women. Aromatase inhibitors (AIs) have been shown to be superior to Tamoxifen in the treatment of ER-positive breast cancer and the prevention of contralateral breast cancer in postmenopausal women with fewer side effects. It is likely that AIs will have a prophylactic role to play in BRCA gene carriers who opt to have prophylactic oophorectomy (PO) and breast surveillance. However, this potential role requires further evaluation through prospective controlled trials, with the inclusion of quality of life issues and skeletal complications as secondary endpoints.Based on the knowledge that the risk of ovarian cancer and mortality is reduced by 50% and 80% respectively in long-term users of oral contraceptive pills (OCPs),62, OCPsThe non-specific action of existing DNA damage reagents poses a problem regarding chemoprevention in BRCA women. Such agents have been found to cause a similar extent of damage in both BRCA1 mutant and wild-type cells. The recent discovery that PARP-1 inhibitors kill BRCA1/2 deficient cells with a high level of specificity subsequently opens up a potential therapy which looks very hopeful for BRCA1 cancer patients. A number of studies have illustrated that BRCA1/2 pre-cancerous cells are very sensitive to PARP inhibition, whilst cancer cells exhibit different responses to PARP inhibition under a variety of different conditions. Furthermore, it has been suggested that these differences in PARP sensitivity can be attributed to distinct phases in BRCA associated tumorigenesis. Work conducted by De Soto & Deng demonstrRisk-reducing surgery in BRCA1/2 gene mutation carriers includes prophylactic bilateral salpingo-oophorectomy (PBSO) and/or prophylactic bilateral mastectomy (PBM). The aim of surgery is to reduce the risk of cancer development and to reduce mortality.Notably, extensive counselling should be offered to all women who consider prophylactic surgery, including a detailed explanation of the cancer risk, complications associated with surgery, aesthetic outcome, quality of life issues, and potential life expectancy gain. The decision to proceed with prophylactic surgery is largely patient-driven depending on whether the patient feels comfortable living with the estimated risk and how she values the psychosexual function of the breast and ovaries. It is observed that many women who undergo BRCA testing use these results to make clinical decisions; those who choose risk-reducing surgeries typically do so within months of receiving BRCA-positive results. Predictors of risk-reducing surgery uptake include age below 60 years, prior breast cancer, and utilisation of another risk-reducing surgery.Since the establishment of the link between breast and ovarian cancers and BRCA1/2 genes mutations, in addition to the increasing availability of genetic testing, research groups were faced with many questions concerning the efficacy and usefulness of risk-reducing surgery. Earlier studies have more keenly focused on decisions concerning whether or when to undergo prophylactic surgery. The magnitude of the potential benefit fundamentally depends on the risk of cancer associated with specific mutations, the prognosis of the tumours in carriers of the mutations, and the extent to which relief of anxiety could result from surgical prophylaxis. These benefits were to be weighed against an array of potential costs, including surgical complications and the potential impacts of mastectomy or oophorectomy on a woman's self-image, as well as on their sexual and reproductive function.There are no randomised controlled trials which have previously examined the potential impacts of PBM on survival; therefore, evidence has been derived from retrospective and short follow-up prospective studies in addition to hypothetical mathematical models with variable estimates.et al., it was concluded that a 30-year-old BRCA1/2 positive woman could prolong her survival by 0.9 years  by having bilateral oophorectomy, 3.4 years (2.7-3.7 years) by having bilateral mastectomy, and 4.3 years (3.6-4.6 years) by having both procedures compared with surveillance alone. Chemoprevention with Tamoxifen and raloxifene was estimated to increase survival by 1.6 years (1.0-2.1 years) and 2.2 years (1.3-2.8 years), respectively. These findings suggest that prophylactic surgery is associated with substantial survival benefits[et al. [Earlier studies have utilised the Markov model, which w benefits,68. Othe benefits. Furthers[et al. ; howeverFurthermore, in the case of carriers of BRCA1/2 mutations who subsequently developed breast cancer, it was found that, with the use of the Markov model and depending on the assumed penetrance of the BRCA mutation, probabilities associated with developing contra lateral breast cancer and ovarian cancer, dying from these cancers, dying from primary breast cancer, and the reduction in cancer incidence and mortality due to prophylactic surgeries and compared with surveillance alone, 30-year-old early-stage breast cancer patients with BRCA mutations showed a gain in life expectancy of approximately 0.4 to 1.3 years from Tamoxifen therapy, 0.2 to 1.8 years from PO, and 0.6 to 2.1 years from prophylactic contralateral mastectomy (PCM). The magnitude of these gains is believed to be least for those women with low-penetrance mutations  and greatest for those with high-penetrance mutations . Notably, older age and poorer prognosis from primary breast cancer further attenuate such gains . UndoubtIn recent years, a few cohort and case control studies have considered the effectiveness of prophylactic surgery. However, it is of great importance to mention that they differ in quality, with some having significant methodological flaws--mainly owing to selection bias and retrospective study design.et al., 483 women with BRCA1/2 mutations were studied. After a mean follow-up of 6.4 years, breast cancer was diagnosed in two (1.9%) of 105 women who had bilateral prophylactic mastectomy, and in 184 (48.7%) of 378 matched controls who did not have the procedure. The group therefore subsequently concludes that BPM reduces the risk of breast cancer in women with BRCA1/2 mutations by approximately 90%[Prophylactic bilateral mastectomy reduces the risk of breast cancer in BRCA mutation carriers by 85%-100%-74. In aately 90%. Similarately 90%.It is accepted that no surgical technique for prophylactic mastectomy removes all breast epithelium; simple mastectomy removes approximately 95%-99% of breast glandular tissue; however, it causes a significant deformity, changes in body image and sexuality, as well as adverse effects on the psychosocial status of the affected woman, but not quality of life. MoreoveSkin sparing mastectomy and immediate breast reconstruction (SSM & IMR) has been proved to be oncologically safe, and is associated with high satisfaction rate and low morbidity,80. MoreSince breast tumours are largely oestrogen-driven, it has been suggested that the hormonal blockade by oophorectomy inhibits the development of breast tumours. Importaet al. found that, during a 3-year follow-up, BPSO was associated with 85% reduction in BRCA1-associated gynaecologic cancer risk and 72% reduction in BRCA2-associated breast cancer risk. Although protection against BRCA1-associated breast cancer and BRCA2-associated gynaecologic cancer was suggested, neither effect reached statistical significance. The authors postulate that the protection conferred by PBSO against breast and gynaecologic cancers may differ between the carriers of BRCA1 and BRCA2 mutations[In a large, retrospective analysis of 551 BRCA carriers, BSO was found to have reduced the risk of ovarian cancer by 96% and breast cancer by 53% at a mean follow-up of 9 years. Similarmutations.Although several studies demonstrate that PBSO decreased the risk of both breast and ovarian cancers in BRCA1/2 mutation carriers-94, theset al. carried out a meta-analysis of ten studies which investigated breast or gynaecologic cancer outcomes in BRCA1/2 mutation carriers who had undergone PBSO. The authors subsequently concluded that PBSO was associated with a statistically significant reduction in breast cancer risk amongst BRCA1/2 mutation carriers. Similar risk reductions were observed in BRCA1 mutation carriers and in BRCA2 mutation carriers. Furthermore, PBSO was associated with a statistically significant reduction in the risk of BRCA1/2-associated ovarian or fallopian tube cancer; however, data were deemed insufficient to obtain separate estimates for ovarian or fallopian tube cancer risk reduction. Moreover, PBSO was strongly associated with reductions in the risk of breast, ovarian, and fallopian tube cancers, and should provide guidance to women in planning cancer risk-reduction strategies[Recently, Rebbeck trategies.Furthermore, in a prospective study with a short-term follow-up, PBSO was associated with a 90% reduction in breast cancer-specific mortality, a 95% reduction in gynaecologic cancer-specific mortality, and a 76% reduction in overall mortality . Such daThis review paper provides an up-to-date analysis of our understanding of the advantages of risk-reducing strategies in BRCA carriers. Although the article summarises the evidence which suggests that different strategies are associated with varying degrees of cancer risk-reduction, such evidence is nevertheless derived from retrospective and short follow-up prospective studies in addition to hypothetical mathematical models. It is known that retrospective studies do not provide the highest level of evidence compared with randomised controlled studies; they are often less robust and usually carry a degree of bias. Furthermore, they tend not to be standardised in relation to confounding factors, such as patients' age, socio-economic class, and other factors.Currently, there are no randomised controlled trials concerned with the demonstration of the potential benefits or harms of prophylactic surgery. This is due to different factors, such as the small number of BRCA mutation carriers, which necessitate multiple centres to participate. Conducting a randomised controlled trial in multiple centres poses potential problems, namely a lack of standardisation, which is the main issue as different surgeons have different surgical risk-reducing techniques. Furthermore, in light of the currently available evidence from retrospective studies, randomisation is deemed unethical, and women may therefore opt for risk-reducing surgery rather than observation.Selecting the most appropriate risk-reducing strategy is not a straightforward task. The impact of risk-reducing strategies on cancer risk, survival, and overall quality of life are the key criteria considered for making a good decision. There is no sole risk-reducing strategy which is able to fully meet all expectations and requirements in an individual woman. On the one hand, non-surgical procedures provide a good body image and quality of life, but may be associated with increased risk of advanced-stage cancer and mortality; on the other hand, surgery ensures a very high protection from cancer but is associated with a number of disadvantages of invasiveness, non-reversibility and surgical morbidity.Based on the available literature, it is deemed appropriate to acknowledge that the clinical management of cancer risk in BRCA1 and BRCA2 mutation carriers is a rather complex and stressful task, which should consider individual patients' expectations and preferences. Such preferences can be informed by accurate knowledge of the risks and benefits of the interventions considered. Available studies confirm the essential role of PBSO in reducing the risk of both breast and ovarian cancer. Furthermore, other options for risk reduction should be considered, such as chemoprevention and risk-reducing mastectomy. AlthougIt is recommended that prophylactic surgery should be performed as soon as possible, simply because of the early development of cancer in BRCA mutation carriers. Although ovarian cancer rarely occurs in premenopausal women, it is advised that BRCA mutation carriers should undergo PBSO immediately after childbearing is complete in order to reduce the risk of early breast cancer development.Prospective randomised controlled trials examining the various interventions in relation to the woman's age and type of mutation are needed. However, the randomisation of BRCA carriers to different arms of trials is an extremely difficult and rather impossible process given the current available evidence.DCIS: Ductal carcinoma in-situ; MRI: Magnetic resonance imaging; SERMs: Selective oestrogen receptor modulators; AIs: Aromatase inhibitors; PO: Prophylactic oophorectomy; OCPs: Oral contraceptive pills; PBSO: Prophylactic bilateral salpingo-oophorectomy; PBM: Prophylactic bilateral mastectomy; PCM: Prophylactic contralateral mastectomy; SSM: Skin sparing mastectomy; IBR: Immediate breast reconstructionThe authors declare that they have no competing interests.MS performed literature search/review and manuscript writing. SB: helped in manuscript writing, KM involved in the concept, manuscript revision and writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6874/10/28/prepub


Arginine is an amino acid that serves as a substrate for the enzymes nitric oxide synthase (NOS) and arginase, leading to synthesis of NO and ornithine, respectively. As such, arginine has the potential to influence diverse fundamental processes in the lung.We used mice deficient in cationic amino acid transporter (CAT) 2 in models of allergic airway inflammation and pulmonary fibrosis.In situ mRNA hybridization demonstrated marked staining of CAT2, predominantly in scattered mononuclear cells. Analysis of allergic airway inflammation and bleomycin-induced inflammation in CAT2-deficient mice revealed that while inflammation was independent of CAT2 expression, bleomycin-induced fibrosis was dependent upon CAT2. Mechanistic analysis revealed that arginase activity in macrophages was partly dependent on CAT2.We report that the arginine transport protein CAT2 was over-expressed in the lung during the induction of allergic airway inflammation. Furthermore, CAT2 mRNA was strongly induced by transgenically over-expressed IL-4, and allergen-induced expression was dependent upon signal-transducer-and-activator-of-transcription (STAT) 6. Taken together, these results identify CAT2 as a regulator of fibrotic responses in the lung. T gondii and develop enhanced fibrosis and granuloma formation in response to S mansoni  probes were hybridized and washed under high-stringency conditions.escribed . In brie32P, and hybridized using standard conditions, as described previously , was liberated with NdeI and MluI, confirmed by sequencing, radiolabelled with eviously .Collagen accumulation was determined by measuring the content of hydroxyproline as previously described . Additio5 cells/100 μl PBS and 22 mM glucose/well of 96-well-plate) were incubated with 14C-arginine  for 18 hours, and 14C-urea present in the cell supernatant was metabolized with urease. Released 14CO2 was collected and measured by scintillation counting. Arginase activity in lysed macrophages was measured using the blood urea nitrogen reagent  according to established techniques [Arginase activity in intact cells  was measured using incorporation of radioactive arginine as previously described . Cells  in the asthmatic lung compared to saline-challenged lungs. We chose to focus on this gene since arginine transported by CAT2 can be metabolized by the arginase and NOS pathways, both of which may have a profound effect on inflammation. Interestingly, microarray analysis revealed very specific expression of CAT2 compared with other CAT genes. For example, the hybridization signals for CAT1 and CAT3 were not detectable in the saline- and allergen-challenged lung (data not shown). We substantiated these findings by Northern blot analysis. In contrast to the saline-challenged control mice which had low levels of CAT2, mice challenged with OVA or Aspergillus had significantly increased levels of CAT2 mRNA . As seen in Figure Since CAT2 was not involved in inflammation, we hypothesized that it would be involved in lung fibrosis. This is based on the following: 1) CAT2 is upregulated in bleomycin-induced pulmonary fibrosis  and 2) aIn order to address the role of CAT2 in collagen deposition, we measured hydroxyproline levels in the lungs of bleomycin-treated mice. As seen in Figure CAT2 may regulate fibrotic processes via arginase activity. Indeed, arginase expression is increased in the lungs of bleomycin-treated mice . Thus, wRecent studies have suggested a role for arginine transport and metabolism in a variety of inflammatory disorders. In this report, we used CAT2-deficient mice to test the role of arginine transport in lung inflammatory conditions, including experimental asthma and bleomycin-induced lung fibrosis. Our studies revealed several novel findings. First, we demonstrate that CAT2 mRNA expression is induced in experimental asthma and that the main cell type expressing it is a mononuclear cell most consistent with alveolar macrophages. Second, we demonstrate that CAT2 is required for bleomycin-induced pulmonary fibrosis. Third, in contrast to the fibrosis data, we show that CAT2 expression is not required for lung inflammation in either allergen-induced or bleomycin-induced models. Finally, we show that CAT2 is partially required for arginase activity in macrophages, which may contribute to the development of fibrosis.Even though CAT2 is induced highly in asthma, it is not required for experimental asthma-induced inflammation. This observation is consistent with our recent finding that arginase is not required for allergic airway inflammation, despite high level of induction . In contSimilarly, the role of CAT2 in fibrosis is context-dependent. Baseline fibrosis is increased in CAT2-deficient mice as measured by the whole lung hydroxyproline assay . InteresThe requirement for CAT2 for arginase activity in macrophages is controversial. Thompson et al.  demonstrIn summary, we have described a pathway (involving CAT2 and arginine homeostasis) not previously examined in the context of lung inflammation and fibrosis. We find that while CAT2 is highly upregulated in experimental asthma, it does not have a role in allergic airway inflammation. Rather, CAT2 is critically important for interstitial lung fibrosis.BALF: bronchoalveolar lavage fluid; CAT: cationic amino acid transporter; NOS: nitric oxide synthase; OVA: ovalbumin; STAT6: signal-transducer-and-activator-of-transcription 6The authors declare that they have no competing interests.KAN, MGC and NZ performed the research; LGE provided critical reagents (CAT2-deficient mice); MER participated in the conception and design of the study and helped revise the manuscript; LGE and MGC helped revise the manuscript; NZ participated in the conception, design and coordination of the study, analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.