Molecularly targeted therapy is relatively new to ovarian cancer despite the unquestionable success with these agents in other solid tumours such as breast and colorectal cancer. Advanced ovarian cancer is chemosensitive and patients can survive several years on treatment. However chemotherapy diminishes in efficacy over time whilst toxicities persist. Newer biological agents that target explicit molecular pathways and lack specific chemotherapy toxicities such as myelosuppression offer the advantage of long-term therapy with a manageable toxicity profile enabling patients to enjoy a good quality of life. In this review we appraise the emerging data on novel targeted therapies in ovarian cancer. We discuss the role of these compounds in the front-line treatment of ovarian cancer and in relapsed disease; and describe how the development of predictive clinical, molecular and imaging biomarkers will define the role of biological agents in the treatment of ovarian cancer. Ovarian cancer is a leading cause of cancer mortality being the most frequent cause of death from gynaecological cancer, and the fourth most frequent cause of death from cancer in women in Europe and the USA. In 2002, there were an estimated 204,000 new cases of ovarian cancer and 125,000 deaths due to this disease worldwide. The incidence rates of ovarian cancer are highest in the Western world, where it is the leading cause of death from gynecological malignancies [The 5 year survival rate of ovarian cancer ranges from 30% to 92%, depending on the stage at diagnosis ,5,6,7,8.Frontline treatment with a platinum-paclitaxel combination is the internationally accepted standard of care in chemo-naïve advanced or recurrent ovarian cancer. At relapse, platinum compounds remain the mainstay of treatment. In platinum-sensitive disease i.e. where the treatment-free interval (TFI) is more than 6 months the response rates can be greater than 50%, but it is only 10–20% for platinum-resistant disease (TFI <6 months) and less for platinum-refractory disease where the disease progresses on treatment. The latter are therefore usually treated with other non-platinum agents, such as liposomal doxorubicin, gemcitabine, topotecan, etoposide and hormonal therapies .A greater understanding of tumour biology and molecular pathways that mediate cancer progression and drug resistance has led to the development of various molecular targeted therapies such as monoclonal antibodies, small molecule receptor tyrosine kinase inhibitors and agents blocking downstream signalling pathways. The development of molecular-directed therapies received a tremendous boost with the confirmation of efficacy of imatinib in GISTs , of bevaAngiogenesis is essential for normal ovarian physiological processes such as follicle maturation and the development of corpus luteum during normal ovulation. Pathological neoangiogenesis on the other hand is a key factor in the growth and development of ovarian tumours. Epithelial ovarian cancer over-expresses vascular endothelial growth factor (VEGF) and other proangiogenic proteins like platelet-derived growth factor (PDGF) and angiopoietin. Increased microvessel density and over-expression of VEGF and its receptor have been correlated with aggressive clinical features, formation of ascites and poor outcome. Conversely, in animal models it has been shown that VEGF blockade inhibits ascites formation and slows ovarian cancer growth ,20,21,22Antiangiogenic therapies inhibit new blood vessel growth, induce endothelial cell apoptosis, block the incorporation of haematopoietic and endothelial progenitor cells into new blood vessels, and normalize the vasculature . These eBevacizumab  is a 149-kDa recombinant humanized monoclonal IgG1 antibody directed against human VEGF. In preclinical models of ovarian cancer, a VEGF-targeted antibody alone had minimal effect on tumour burden, but markedly decreased ascites . HoweverSingle agent bevacizumab has been evaluated in the treatment of patients with recurrent ovarian carcinoma in two phase II clinical trials at a dose of 15 mg/kg administered every 3 weeks in patients who had received one to three previous chemotherapeutic regimens ,32, TablCytotoxic chemotherapy is conventionally administered at a dose close to its maximum tolerated dose, with breaks to allow the recovery of normal tissues. Chemotherapy at this dose does kill proliferating vascular endothelial cells, but recovery happens rapidly. In animal models, lower doses of chemotherapy for example, paclitaxel, vinorelbine and cyclophosphamide given frequently (metronomic dosing) target proliferating endothelial cells leading to their apoptosis with few side effects and no significant direct tumour cytotoxicity ,36,37. et al. [Metronomic docetaxel chemotherapy in combination with AEE788, (a combined EGFR and VEGFR inhibitor), has shown encouraging activity in an orthotopic mouse model of ovarian cancer utilising a cell line resistant to conventional chemotherapy dosing . This apet al.  a responet al. [vs. 10%, p = 0.07) at the cost of increased toxicity. Grade 3–5 toxicities occurred in 15 (24%) patients, including hypertension (7%), gastrointestinal perforations (7%), and chylous ascites (5%). Gastrointestinal perforations occurred in heavily pretreated patients who were responding to therapy. Development of chylous ascites and gastrointestinal perforations appeared to correlate with tumor response. The overall response rate was 36%, with stable disease in 40%. The GOG 213 study aims to recruit 1,600 patients in a bifactorial design of carboplatin and paclitaxel alone or in combination with bevacizumab followed by bevacizumab and secondary cytoreductive surgery in platinum-sensitive, recurrent ovarian, fallopian tube and primary peritoneal carcinoma.A multi-institutional retrospective evaluation of factors predictive of toxicity and efficacy of bevacizumab in patients with recurrent ovarian cancer was performed by Wright et al. . Single-In chemotherapy-naïve patients with advanced ovarian carcinoma, carboplatin/paclitaxel and bevacizumab produced a response rate of 75%–80% with acceptable toxicity ,41, TablOne of the most effective ways to block the VEGF signalling pathway is to prevent VEGF from binding to its normal receptors by administering decoy VEGF receptors. Aflibercept (VEGF Trap), is one such soluble decoy receptor. It is a fusion protein containing the VEGF-binding domains of both VEGFR-1 and -2 linked through the Fc region of human IgG1 and is a potent inhibitor of VEGFA. In a Phase II double-blind study of patients with platinum-resistant ovarian cancer, 162 patients were treated at two dose levels of 2 mg/kg or 4 mg/kg every 2 weeks. Independently assessed response rates were 11%. Side-effects were reassuringly typical of this class of drugs with hypertension being the most common . HoweverActivated receptor tyrosine kinases (TKIs) phosphorylate numerous signalling molecules activating downstream signal transduction pathways leading to tumour cell proliferation and survival. These phosphorylation dependent mechanisms are essential for promoting the activity of growth factors like VEGF and PDGF. Blocking phosphorylation by targeting the intracellular component of tyrosine kinase thereby inhibiting the biological activity of VEGF is therefore an effective antitumour strategy.Promising results from several phase II trials investigating single agent small molecule TKIs, that target VEGFR in relapsed ovarian cancer are summarized in Vertical blockade by inhibition at different points in the VEGF signalling pathway has been mooted as a strategy to enhance efficacy. This was explored in a phase I study of sorafenib and bevacizumab which demonstrated durable partial disease responses in 6 of 13 ovarian cancer patients recruited. Unexpectedly, toxicity was substantially higher than that with single-agent anti-VEGF therapy, with two thirds of the patients developing hypertension and 79% incidence of grade 1–3 hand–foot syndrome. Enteral fistulae were also seen in 2 of the 13 ovarian cancer patients in the study . EGFR and VEGF share common downstream signalling pathways. VEGF is down-regulated by EGFR inhibition, and conversely blockade of VEGF may also inhibit EGFR autocrine signalling . Dual blAn approach that combines bevacizumab and a Vascular Disrupting Agents (VDA) was explored as preclinical data provide evidence for synergy between a VDA, which induces a surge in VEGF-stimulated circulating endothelial progenitor cells, and bevacizumab, which suppresses this VDA-induced effect. No additive toxicity was seen in phase I study of bevacizumab and a VDA combretastatin 4A phosphate ,56.Vascular disrupting agents (VDAs) target endothelial cells and pericytes of the already established tumour vasculature, resulting in tumour ischemia and necrosis. VDAs have been divided into two types: ligand-directed VDAs and small molecules. Ligand-directed VDAs consist of targeting and effector moieties that are linked together. Their clinical efficacy appears limited because of cost and a lack of specificity and toxicity. Small molecules include two classes: the synthetic flavonoids, which work through induction of local cytokine production, and the tubulin-binding agents . CombretBevacizumab is associated with several well recognized toxicities including hypertension, proteinuria and minor bleeding. More serious, but rarer events, are arterial thrombosis, impaired wound healing, major bleeding, gastrointestinal perforation (GIP), reversible posterior leukoencephalopathy and tracheo-oesophageal fistulae. A metaanalysis of all prospective randomized studies of bevacizumab in solid tumors showed the risk of GIP to be 0.9% with a mortality of 21.7% . IncidenHypertension is a side-effect noted with bevacizumab and other small molecule TKIs. A metaanalysis of randomized controlled trials with patients receiving bevacizumab showed a relative risk of 3.0 for hypertension at low doses (2.5–7.5 mg/kg) and 7.5 for high doses (10–15 mg/kg); and for proteinuria 1.4 at low doses and 1.6 for higher doses . There iThe epidermal growth factor receptor  is a tyrosine kinase receptor of the ErbB family (ErbB1-4) that is abnormally activated in many epithelial tumours. Receptor activation leads to recruitment and phosphorylation of several downstream intracellular substrates, leading to mitogenic signalling and other tumour-promoting cellular activities. In human tumours, receptor overexpression correlates with a more aggressive clinical course. Taken together, these observations indicate that the EGFR is a promising target for cancer therapy. The two main classes of compounds specifically targeting EGFR include low-molecular weight Tyrosine Kinase Inhibitors (TKIs) and anti-EGFR monoclonal antibodies (MAbs) and these are at different stages of development, summarized in EGFR is overexpressed in a high proportion of ovarian carcinomas ,65. FollBiomarker driven anti-EGFR therapy is now established in metastatic colorectal cancer with the discovery that tumours with mutated KRAS do not respond to anti-EGFR therapy . This, aPoly (ADP-ribose) polymerase (PARP) is a key enzyme involved in the surveillance and maintenance of genomic integrity (reviewed in ). It funOlaparib  is an potent, orally active, small-molecule PARP inhibitor which inhibits PARP1 and PARP2 with a mean IC50 of 2 nM . PreclinThe Phase I trial of olaparib was inclusive of patients with a wide range of drug-resistant cancers for the escalation phase . After tThe results of the Phase II trial, presented at ASCO 2009, primarily aimed to test the efficacy of olaparib in confirmed BRCA1/BRCA2 carriers with advanced chemotherapy-refractory ovarian cancer . In thisPARP inhibitors may also have a role in sporadic ovarian cancers with homologous recombination defects, which might result from nonfunctional proteins rendering these cells sensitive to PARP inhibition . These sThe PI3K-PTEN-Akt-mTOR pathway is implicated in multiple biological processes including cell proliferation, protein translation, autophagy, cell metabolism, cell proliferation, angiogenesis and prevention of apoptosis. Alterations of this signalling pathway are well documented in cancer and are believed to result in activation of downstream signalling pathways that drive oncogenesis . For thiPI3K has at least eight isoforms of catalytic subunit and seven regulatory subunits. The PI3K proteins implicated in tumorigenesis belong to a subclass called 1A. Genetic amplification of PIK3CA, which encodes p110α catalytic subunit of PI3K  have been observed in approximately 40% of ovarian cancers . TransfoPI3K signalling is negatively regulated by the protein phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome ten). PTEN can be considered as a tumour suppressor, preventing Akt activation through the dephosphorylation of a PI3K lipid signalling product. The elimination of PTEN activity  can lead to oncogenesis through activation of Akt, which in turn upregulates mTOR activity. Therefore, cancer cells deficient in PTEN are an attractive target for mTOR inhibitors. PTEN aberrations have been implicated in ovarian cancers, especially those with an endometrioid histology , and losAkt is an intracellular serine/threonine kinase which is a critical regulatory switch, and is believed to phosphorylate over 9,000 proteins (reviewed in ). ConseqClinical trials of PI3K and Akt inhibitors have only recently commenced. However, successful modulation of the PI3K-PTEN-Akt-mTOR pathway for anticancer therapy has been demonstrated with the mTOR inhibitor rapamycin and its analogues temsirolimus , everolimus  and deforolimus  (reviewed in ) While cCa-125 is a surface glycoprotein antigen elevated in more than 95% of patients with stage III and IV ovarian cancers . While CAn anti-EPCAM and anti-CD3 antibody currently undergoing clinical evaluation is catumaxomab  which is known to kill tumour cells within ascitic fluid by activating T cells and accessory cells. A randomised study of catumaxomab in patients with malignant ascites (50% of which had ovarian cancer) demonstrated that patients that received intraperitoneal infusions of catumaxomab had a significant prolongation of puncture-free survival and puncture-free time . Side efAntifolate agents are also in clinical development. The folate transporter α-FR is overexpressed in 90% of ovarian cancers compared with normal tissue, possibly promoting growth by increasing folate availability . The degThe emergence of targeted therapies offers the potential of less (different) toxicities and improved efficacy, both as single agents based on specific molecular aberrations, and in combination with conventional cytotoxics to overcome drug resistance. One key challenge is determining the best placement of targeted therapies within the armament of existing treatments: first-line, maintenance / consolidation therapy, at relapse / remission, in combination. Traditionally, novel agents are often combined with existing cytotoxics, such as in the ICON-7 trial in which patients with newly diagnosed ovarian epithelial, fallopian tube or primary peritoneal cancer receive carboplatin and paclitaxel chemotherapy with or without bevacizumab. Likewise the ICON 6 study is investigating the efficacy of cediranib, an oral VEGF receptor tyrosine kinase inhibitor with chemotherapy in relapsed platinum-sensitive ovarian cancer. Combining targeted therapies poses the additional question of whether it is most efficacious to inhibit different signalling pathways  or different molecules within the same pathway to counteract pathway redundancy . The pitfalls of combining targeted agents have been demonstrated in trials combining bevacizumab and sorafenib , and bevIn future it is unlikely that ovarian cancer will be treated as a single entity, but will instead be based on molecular characterisation. From the evidence presented here, patients with homologous recombination-defective tumours could be considered for PARP inhibitors and endometrioid subtypes could be candidates for PI3K-PTEN-Akt-mTOR therapies. However, it is unlikely that histological subtype alone will be sufficient to account for tumour molecular heterogeneity. Molecular profiling of the tumour and normal tissues will enable better understanding of the effects of inhibiting the target in tumour and host tissue. Additionally, genetic polymorphisms may significantly affect drug distribution and tolerance and this is being harnessed in the field of pharmacogenomics. This will enable patients to be selected for a treatment to which they are most likely to respond . In order to better understand molecular aberrations in ovarian cancer, clinical trials will need to mandate the collection of high quality reference material before, during and after treatment e.g. at disease relapse. This should not detract from clinical parameters and will permit correlation with toxicity and response evaluation. In the future, the ‘diagnostic biopsy’ will be characterised and the patient ‘profiled’ to select a treatment to which the patient will most likely respond. This archived material can then be compared with tumour tissue at disease relapse, to identify stem cells that may account for a resistant population of tumour cells .Traditionally, tumour biopsies have been the gold-standard reference material used to predict responsiveness to a therapy. However, difficulty in obtaining samples has led to the development of surrogate end-points and the inclusion of pharmacodynamic endpoints in clinical trials to establish proof of mechanism. Optimal development requires a reliable, validated assay to evaluate target inhibition and a knowledge of the predicted effects of target inhibition, e.g., tumour shrinkage, cytostasis, decreased vasculature. Extensive preclinical studies, both in vitro and in vivo are required to expedite drug development so that clinical trials can be targeted to patients who carry the relevant molecular aberrations. Traditional phase I end-points such as toxicity may need to be replaced in order to define the biologically effective dose with molecularly targeted anticancer agents e.g. plasma drug concentration or target inhibition in surrogate tissue. Primary end-points such as objective tumour response by anatomical imaging  commonly used for conventional cytotoxic drugs may not detect cytostatic effects and functional imaging such as DCE-MRI and FDG-PET may be more useful biomarkers. The co-development of novel targeted drugs with biomarkers is essential as new treatments are associated with high costs, and many fail unpredictably in late clinical trials (reviewed in ). Compar


Systemic treatment for triple negative breast cancer (TNBC: negative for the expression of estrogen receptor and progesterone receptor and HER2 amplification) has been limited to chemotherapy options. Neoadjuvant chemotherapy induces tumor shrinkage and improves the surgical outcomes of patients with locally advanced disease and also identifies those at high risk of disease relapse despite today's standard of care. By using pathologic complete response as a surrogate endpoint, novel treatment strategies can be efficiently assessed. Tissue analysis in the neoadjuvant setting is also an important research tool for the identification of chemotherapy resistance mechanisms and new therapeutic targets. In this paper, we review data on completed and ongoing neoadjuvant clinical trials in patients with TNBC and discuss treatment controversies that face clinicians and researchers when neoadjuvant chemotherapy is employed. Neoadjuvant chemotherapy, also known as preoperative or primary systemic therapy, is an option for patients with breast cancers who require cytotoxic chemotherapy. It was initially used for patients with locally advanced inoperable breast cancers . SubsequTriple negative breast cancer (TNBC) is defined clinically by the absence of estrogen receptor (ER), progesterone receptor (PgR), and HER2/neu overexpression and encompasses a molecularly diverse group of diseases. As TNBC lacks a clearly defined therapeutic target, patients receive chemotherapy for their systemic management. Since chemotherapy-resistant TNBC carries a particularly poor prognosis, the identification of the mechanism of chemoresistance and therapeutic advances are critical. There is therefore a particularly strong rationale for clinical research in this setting.Breast cancer is subdivided into at least five major intrinsic subtypes, including luminal A, luminal B, HER2-enriched, basal-like, and claudin-low breast cancer . The majP = 0.042) with third generation, taxane containing regimens, as compared to mesenchymal-like (31%) or luminal androgen receptor (14%) subtypes [In an effort to identify TNBC-specific molecular subtypes, Lehmann et al. analyzed the gene expression profile of 587 TNBC cases . Six subsubtypes . In addisubtypes . Mesenchsubtypes . This fisubtypes . InteresIt is likely that the classification of TNBC will continue to evolve and we envision the development of treatment-directed classification to also include necessary markers by either genomic or proteomic analysis. Neoadjuvant setting provides a platform for proof of concept studies in this regard. A consensus conference on TNBC definitions, similar to historic efforts in lymphoma and leukemia, should be considered to facilitate subset specific clinical trials.P = 0.69). Similarly, the GeparTrio study reported by Huober et al. showed a pCR rate of 39% in the TNBC patient subset on this trial which sought to determine the effect on pCR of switching neoadjuvant chemotherapy depending on mid-course response [ While several clinical trials involving traditional chemotherapy, or biologics have recently been launched in an attempt to improve the outcome of patients with TNBC, most neoadjuvant studies have not restricted entry criteria to only patients with TNBC and so the attempt to define treatment standards has required retrospective subset analysis, risking underpowered investigation. Nonetheless a large proportion (41%) of the study population in NSABP protocol B-40 was classified as having TNBC . The goaresponse . PatientP < 0.05) with the addition of carboplatin in the TNBC group but not in the HER2+ breast cancer group  . The Cancer and Leukemia Group B (CALGB) 40603 is evaluating the addition of carboplatin and/or bevacizumab to standard chemotherapy in the neoadjuvant setting for TNBC [ As sporadic TNBC has clinical and molecular similarities to BRCA-1-associated breast cancers, there has been significant interest in using platinum compounds in TNBC. BRCA-1-associated breast cancers are sensitive to these agents since double-strand DNA breaks cannot be correctly repaired in cells deficient in homologous recombination repair mechanisms. Several neoadjuvant trials have therefore evaluated platinum agents in TNBC patients . Alba etfor TNBC . The chaP = 0.003). These findings were not confirmed in the other large neoadjuvant study, NSABP B-40 [Different biologics such as antiangiogenic agents, poly-ADP ribose polymerase (PARP) inhibitors, and other small molecule inhibitors are being evaluated in patients with TNBC. As a higher level of vascular endothelial growth factor (VEGF) has been detected in TNBC , the VEGABP B-40 . Here, a PARP enzymes mediate repair of single-strand DNA breaks through base excision repair mechanism. Loss of PARP enzymes will therefore lead to accumulation of single-strand breaks which under normal circumstances ought to be repaired by the homologous recombination pathway. Since the homologous recombination pathway is deficient in BRCA-associated breast cancers, PARP inhibition leads to “synthetic lethality” in these cancers. As TNBC have clinicopathologic similarities to BRCA associated breast cancers, PARP inhibitors are attractive treatments for this group of patients. Olaparib has been investigated in patients with advanced TNBC- or BRCA-associated breast cancer with mixed results –39. OtheOther features of TNBC such as activation of the PI3 K/AKT/mTOR pathway and notch signaling and overexpression of EGFR and c-KIT continue to be exploited for treatment opportunities , 28 Tab.Achievement of pCR is highly predictive of long-term outcome for patients with TNBC despite the various definitions of pCR applied in individual studies , 40–42, TNBC is associated with a significantly higher rate of pCR to standard neoadjuvant compared to ER+ HER2− disease. The rate of pCR approximates 20% or higher for TNBC in various studies , 40, 41.NCT01074970) who had Miller-Payne response in the breast of 0–25 or RCB classification of II or III following neoadjuvant chemotherapy. In addition to pCR, quantitation of residual disease burden in the breast and lymph nodes, including the use of residual cancer burden (RCB)  and MillThe induction of a pCR in response to neoadjuvant chemotherapy effectively categorizes heterogeneous TNBC into high- and low-risk groups. Unfortunately, there has not been any effective therapy for those with chemotherapy-resistant disease. There has been significant interest in analyzing residual tumors in order to uncover molecular aberrations as potential therapeutic targets. Balko et al. presented an analysis with targeted next-generation sequencing (NGS) of 182 oncogenes and tumor suppressors and gene expression profiling of residual breast cancer after neoadjuvant chemotherapy in 102 patients with TNBC in the recent San Antonio Breast Cancer Symposium . Eighty-Improvement in the rate of pCR has been considered an accepted endpoint for neoadjuvant clinical trials in patients with TNBC so that drugs could be tested efficiently. The ability to obtain sufficient high-quality tumor material before and after therapy makes it possible to correlate biomarkers with pathologic response. Furthermore, specific biomarkers could be incorporated in the trial design as an eligibility criteria or stratification factor to enhance the power to address specific biomarker hypothesis. To facilitate early access of potentially active drugs for patients with high-risk disease, the Food and Drug Administration (FDA) has recently outlined a pathway for accelerated drug approval based on pCR from neoadjuvant trials. However, disease-free survival (DFS) and overall survival (OS) remain the standard endpoints for eventual drug approval. Large neoadjuvant trials that are powered for these endpoints may be preferred. Alternatively, adjuvant trials could be conducted while the drug receives accelerated approval so that the DFS and OS endpoints could be assessed.The data currently available on neoadjuvant chemotherapy in primary breast cancers leaves many questions unanswered. For instance, what is the role of further non cross-resistant systemic therapy in TNBC patients who do not respond to initial neoadjuvant chemotherapy? In the aforementioned GeparTrio study , 53, patAt the other end of the response spectrum questions are beginning to be asked as to whether a subset of TNBCs who achieve pCR do not require the full complement of local treatments . The consideration to withhold definitive surgery from any patient who achieves such a clinical or radiological response to neoadjuvant chemotherapy is certainly premature at this time considering that our current tools for assessing response to neoadjuvant chemotherapy are very insensitive to microscopic but clinically relevant residual disease. A small European study compared the accuracy of clinical examination, mammography, ultrasonography, and dynamic MRI in predicting response to neoadjuvant chemotherapy . All fifAnother key question concerns the best timing of sentinel lymph nodal biopsy (SLNB) in patients undergoing neoadjuvant chemotherapy. The benefits and disadvantages of performing this procedure either before or after chemotherapy are summarized in  Neoadjuvant chemotherapy is now a standard of care for a subgroup of patients with TNBC, particularly those with clinical stage 2B or 3 disease, but it could be reasonably argued that any patient with a biopsy that shows invasive disease would be eligible. This approach promotes breast conservation and provides an important platform for translational research. pCR is an appropriate surrogate for measuring long-term clinical outcome in TNBC. Although pCR rates to standard chemotherapy are higher in TNBC compared to HR positive breast cancer, the large proportion of patients with TNBC that do not achieve pCR presents a therapeutic challenge. Further studies are therefore critical to identify predictors of resistant population so that mechanism-based interventions could be designed in the neoadjuvant setting to improve pCR rates and long-term outcome. Studies are also needed to address ongoing controversies and debates including the use of axillary surgery and the role of postoperative regional radiotherapy  in patients with pCR.


Cancer stem cells represent a small number of pluripotent and self-renewing cells within a tumor, which are resistant to conventional chemotherapy and are responsible for tumor initiation and maintenance. The Wnt/β-catenin signaling pathway drives stem cell self-renewal and is involved in the pathogenesis of various types of cancer. Aberrant activation of the Wnt signaling pathway in normal stem cells can promote their transformation into cancer stem cells ,2. Thus,Salinomycin, an antibiotic potassium ionophore, is the first drug reported to act as a selective breast cancer stem cell inhibitor. It kills breast cancer stem cells at least 100 times more effectively than paclitaxel in mice, but is relatively non-toxic to normal stem cells . The mecA successful anticancer therapeutic regime should eliminate both the differentiated cancer cells and the cancer stem cell population. Classical cytotoxic agents may deplete the bulk of a cancer but not the inherently chemo-resistant cancer stem cells, which ultimately recur and metastasize. The molecularly targeted agents act on aberrant molecular pathways associated with tumor development and progression, and have been successfully introduced to treat patients with various cancers . These d


Patients after solid organ transplantation (SOT) carry a substantially increased risk to develop malignant lymphomas. This is in part due to the immunosuppression required to maintain the function of the organ graft. Depending on the transplanted organ, up to 15% of pediatric transplant recipients acquire posttransplant lymphoproliferative disease (PTLD), and eventually 20% of those succumb to the disease. Early diagnosis of PTLD is often hampered by the unspecific symptoms and the difficult differential diagnosis, which includes atypical infections as well as graft rejection. Treatment of PTLD is limited by the high vulnerability towards antineoplastic chemotherapy in transplanted children. However, new treatment strategies and especially the introduction of the monoclonal anti-CD20 antibody rituximab have dramatically improved outcomes of PTLD. This review discusses risk factors for the development of PTLD in children, summarizes current approaches to therapy, and gives an outlook on developing new treatment modalities like targeted therapy with virus-specific T cells. Finally, monitoring strategies are evaluated. Progress in solid organ transplantation (SOT) dramatically improved the prognosis for children and adolescents with hereditary or acquired terminal organ failure. Immunosuppressive induction and maintenance regimens were instituted to prevent organ graft rejection by the recipient's immune system. On the downside of pharmacological immunosuppression, a decreased immunological surveillance of infections and malignancies is observed. Pediatric and adolescent patients after SOT carry an increased risk of cancer development, which is estimated to exceed the normal population's up to 45-fold, depending on the type of cancer . The mosThis review focuses on special characteristics of pathogenesis, treatment, and prognosis of PTLD in children and adolescents after SOT.Pathophysiology of PTLD is only partially understood, and its etiology is most probably multicausal. Despite all uncertainties, EBV infections and transplant-related immunosuppression are unquestioned elements of posttransplant lymphomagenesis.EBV is a human oncovirus belonging to the group of gammaherpesviruses. Primary infection with EBV usually occurs during childhood or adolescence, and by the age of 30, more than 90% of the population have become seropositive . Directly after B-cell infection, EBV establishes a nonproductive (“latent”) infection that is divided into four types (latency type 0 to 3) characterized by distinct viral gene expression profiles . Upon spIn vitro EBV infection of B cells results in the outgrowth of immortalized lymphoblastoid B-cell lines (LCLs), which express the latency type 3 program. This “growth program” is characterized by the expression of nine proteins: three latent membrane proteins (LMPs) and six EBV-associated nuclear antigens (EBNAs). These mimic external growth signals (LMP1 and LMP2) or directly regulate gene expression , thereby driving the infected cell into proliferation , 2117, 2P = 0.222) , 1510, 1T-cell depleting antibodies like antithymocyte globulin (ATG) or muromonab-CD3 (OKT3) as well as interleukin-2 (IL-2) receptor specific antibodies like basiliximab or daclizumab are widely used to induce graft tolerance, but their impact on PTLD development is difficult to estimate . Best evAlthough the influence of immunosuppression in the pathogenesis of PTLD is widely accepted, there is no consensus about the impact of a single agent. Differences between CSA and tacrolimus have been discussed in several studies , 22, 36.Mycophenolate mofetil (MMF) is virtually always used in combination with other immunosuppressive drugs, hampering the possibility to analyze its influence separately. Despite these limitations, the addition of MMF does not seem to increase the risk of PTLD , 37, 38.Protective effects were proposed for immunosuppressive drugs belonging to the group of mammalian target of rapamycin inhibitors (mTOR-I), because mTOR signaling pathways are activated in many PTLD cases , 40. NevSince symptoms of PTLD are often unspecific, diagnosis of PTLD requires cell and/or tissue sampling for histopathologic examination. Per definition, every lymphoid malignancy arising after transplantation is classified as PTLD. The histological characteristics are not specific, therefore knowledge about the transplant history is essential for the pathologist to differentiate PTLD from other types of lymphoproliferation. The 2008 WHO classification for lymphoid malignancies  divides Early lesions show oligo- or polyclonal proliferations of EBV-positive B cells while the underlying tissue architecture is preserved. The B cells may either have a predominantly immunoblastic phenotype (infectious mononucleosis like early lesion PTLD) or a plasma-cell phenotype (plasmacytic hyperplasia early lesion PTLD). Like early lesions, polymorphic PTLDs demonstrate oligo- or polyclonal B-cell proliferations, but here, the infiltrating cells destroy the original architecture of the host tissue. While polymorphic PTLD can easily be differentiated from early lesions in lymph nodes, this can be very difficult in extranodal PTLD .All PTLDs fulfilling the histopathologic criteria of “classical” non-Hodgkin's lymphoma (NHL) are diagnosed according to the classification of nontransplant associated lymphomas. Within pediatric registry studies, monomorphic PTLDs account for 35–83% of all cases , 15. TheClassical Hodgkin's disease and Hodgkin-like PTLD also belong to monomorphic PTLDs, but due to their special histological and clinical features, they represent a separate group within the WHO classification. The two basic principles of current standard PTLD treatments are reconstitution of anti-EBV/antitumor immune responses, and, if this is not sufficient, antineoplastic immuno-/chemotherapy (+/− radiotherapy). Both approaches are limited by the patients' pre-existing condition: reconstitution of immune responses carries the risk of transplant rejection, which may be intolerable in some circumstances. On the other hand, the patients may be unable to tolerate the side effects of chemotherapy or radiotherapy due to their medical history.Reduction of immunosuppressive drugs (RIS) is a key component of PTLD therapy in every patient. It may be sufficient to induce complete remission (CR) as a single therapeutic strategy in some patients . HoweverCurrent guidelines recommend RIS wherever possible, but due to heterogenous strategies, the documented effectiveness of RIS still varies between studies. While some retrospective studies report CR in up to 50% of the patients , the onlHowever, when RIS is given to PTLD patients, very close monitoring is mandatory to recognize progressive disease and/or rejection as early as possible , 51. In in vitro [In vivo, encouraging response after a conversion from calcineurin inhibitors to mTOR-I has been reported [mTOR-inhibitors like rapamycin/sirolimus and everolimus are currently evaluated in small clinical trials for their therapeutic effect in hematologic malignancies, and some promising data have been published . mTOR siin vitro . In vivoreported . A detaireported . TherefoOften, modification of immunosuppression will not be sufficient to induce long-term complete remission of PTLD. Cytoreductive drugs (antibodies and/or chemotherapy) are necessary to substantially control PTLD activity in most patients. After the inclusion of the anti-CD20 antibody rituximab into standard regimens, outcome of PTLD treatment improved drastically . A 2-yeaAfter documentation of favorable responses to rituximab also in pediatric PTLD , 62, ritAnthracycline-based chemotherapy ) in combination with rituximab is the standard of care for adult patients with PTLD that are able to tolerate the toxicity associated with this regimen , 65, 66.In children, a recent prospective phase II clinical trial on treatment of PTLD combined rituximab (375 mg/m² weekly for six consecutive weeks) with simultaneous low-dose chemotherapy (six three-weekly courses of cyclophosphamide 600 mg/m² at day 1 together with prednisone 1 mg/kg/d day 1–5) . Hereby,de novo NHL protocols in case of primary treatment failure . Allogeneic hematopoietic stem cell transplantation has been reported as a salvage therapy for PTLD in few case reports , but theThere is very limited data on the significance of radiation therapy in the treatment of pediatric PTLD. In first-line treatment, involved field radiation in Hodgkin's/Hodgkin-like PTLD and cranial irradiation for CNS-PTLD may represent curative treatment elements. However, radiotherapy in pediatric PTLD patients is usually considered only in second-line treatment, both as part of salvage concepts and in palliative care situations. Burkitt's or Burkitt-like PTLD is more aggressive compared to “classical” DLBCL-type PTLD , 70, 71.T-cell PTLD is a rare entity , especiade novo Hodgkin's lymphoma. Hodgkin or Hodkgin-like PTLD accounts for approximately 3–9% of pediatric PTLDs , 15 and Treatment for PTLD affecting the central nervous system (CNS) is difficult, because most drugs used in standard therapy  do not sufficiently cross the blood-brain barrier. Therapeutic attempts with both high-dose intravenous rituximab  and intrAssuming that active EBV replication contributes to the pathogenesis of EBV-positive PTLD, ganciclovir  or cidofTo sensitize EBV-positive PTLD to antiviral drugs, experimental approaches aim at inducing lytic replication of EBV in latently infected cells. Within the course of the lytic cycle, the viral kinase is expressed, which can induce ganciclovir's cytotoxic activity. A clinical phase I/II trial using ganciclovir together with arginine butyrate as inductor of lytic replication activity resulted in CR in a reasonable number of patients with refractory EBV-positive lymphoma .Anecdotal responses to antiviral treatment with foscarnet, a drug that does not require activation by viral kinases, have been reported in adult patients . The sigex vivo expanded EBV-CTLs [EBV proteins expressed in a high proportion of pediatric PTLD tumors are potential targets for tumor-specific immunotherapy via adoptive transfer of virus-specific cytotoxic T-lymphocytes (EBV-CTLs). An overview of published clinical trials is given in EBV-CTLs –88 or EBEBV-CTLs , 90. Witex vivo expanded EBV-CTLs for clinical use by repetitive antigenic stimulation is very laborious and expensive and therefore performed in only few institutions [Unfortunately, the production of itutions . The proitutions . Availability of EBV-CTL is not only limited by labour intensity and costs but also by the fact that the production has to follow the standards of good manufacturing practice (GMP) for open cell culture processes. Therefore, more rapid and easier strategies for the generation of EBV-CTLs are currently being developed. They aim at direct isolation of EBV-CTLs from donor-derived peripheral blood mononuclear cells (PBMC) by labeling with magnetic beads and subsequent purification via magnetic columns. Specific labeling of EBV-CTL is either achieved by the use of EBV-epitope major histocompatibility complex (MHC) class I multimeres  or by cyThe prognosis of PTLD in pediatric patients with PTLD is better than in adult patients. In two prospective studies the 2-year overall survival was 73% in the prerituximab era  and 83% Several efforts have been taken to define parameters estimating the risk of PTLD development and warranting preemptive measures. Serial monitoring of EBV DNA load in the peripheral blood by polymerase chain reaction (PCR) has been proposed as a diagnostic tool to identify patients at high risk of PTLD development. Both the magnitude and the duration of EBV detection in peripheral blood were suggested as critical parameters in PTLD risk estimation. Data from several retrospective analyses point towards a heterogenic picture: pediatric HTx patients with chronic high load (CHL) seemed to be at an increased risk for PTLD development , 104, whDifferent methods of EBV load monitoring are currently used in different laboratories, and the lack of standardization makes it difficult to compare results between centers and draw specific conclusions from individual cut-off values. There seems to be a difference between EBV DNA load in serum and whole blood samples. Stevens et al. demonstrated that early PTLD detection can be achieved in Lung-Tx patients by measuring EBV DNA in the cellular blood compartment, while parallel serum samples were all below the cut-off value , most liMonitoring of cellular immune responses to EBV in addition to EBV DNA load in peripheral blood was suggested to identify patients at risk for PTLD early after transplantation . In our Several serological markers have been evaluated as surrogate markers to predict PTLD development. EBV induces the expression of CD30 on target cells, and soluble CD30 (sCD30) secreted into the serum has been found elevated in patients with PTLD ; however18FDG-PET scan) may prove useful to individually tailor treatment in patients with residual lesions [For monitoring of response to PTLD treatment, clinical and radiologic examinations  still represent the gold standard. Techniques quantifying metabolic activity of morphologic lesions (e.g.,  lesions  similar  lesions  or CXCL1 lesions  may alloIn patients after successful treatment of PTLD monitoring should be performed as suggested by guidelines for lymphomas in nonimmunocompromised persons. The followup is based on regular clinical and radiologic evaluation of remission status. Redetection of EBV in peripheral blood of patients treated with rituximab does not seem to predict disease relapse but may rather be a surrogate of normal B-cell recovery , 117. WhThe ultimate goal will be to completely abrogate PTLD development by effective preventive measures. Some measures have been suggested in high-risk patients, but all require further confirmation until they can be considered standard of care in SOT recipients.Opelz and colleagues analyzed the effect of CMV immunoglobulin and antiviral drugs in more than 42,000 renal transplant recipients . None ofAdoptively transferred EBV-specific T cells were not only shown to have therapeutic effects in manifest PTLD but also to prevent PTLD when given to high risk patients prophylactically after stem cell transplantation  or SOT . HoweverStrategies of active immunization against EBV prior to SOT have been advocated to avoid subsequent EBV primary infection, latent infection, and putative cancer development. In a phase II clinical trial a recombinant DNA vaccine targeting the EBV lytic cycle glycoprotein gp350 reduced the incidence of infectious mononucleosis by 78% in healthy young adults . HoweverSince the first description of PTLD in 1969 , consideAt first, many previous diagnostic and therapeutic attempts have been hampered by the pronounced heterogeneity and relative rarity of the disease. We increasingly understand that there are actually several separate disease entities summarized under the diagnosis of “PTLD.” A better definition of these entities and tailoring of specific treatment schemes are warranted. This can only be achieved by multicentre and international transplant and/or PTLD registries and research collaborations. Ongoing or recently published prospective trials such as Ped-PTLD or the study by Children's Oncology Group  are impoSecondly, new insights into cellular and molecular mechanism need to be better incorporated into clinical research projects. So far, primary diagnostics, risk assessment, and response evaluation mainly rely on clinical parameters. New approaches such as sequencing studies and gene expression profiles  should h


Altered metabolism represents one of the oldest hallmarks associated with cancer. The aberrant metabolic profile deals primarily with the well-known switch of transformed cells towards aerobic glycolysis from mitochondrial oxidative phosphorylation. In addition, it deals with the exacerbation of several biosynthetic pathways interconnected with the increased glycolytic flux, eventually conferring selective metabolic advantages.The distinctive and ubiquitous nature of this altered metabolic profile expressed by cancer cells from various origins or tissues turns the modulation/reversion of these aberrations as an amenable strategy for new anticancer therapies. Despite Otto Warburg's pioneering observations, targeting cancer cell metabolism for therapeutic purposes still remains theoretical. Several factors have contributed to this situation. Primarily, for many years, cancer has been treated as a homogeneous and too simplistic cellular system, where the impairment of proliferation/viability of the most abundant differentiated cancer cells has been extensively pursued. This approach, however, has not been successful in preventing tumour relapse. Cancer is nowadays considered a complex network, including cancer cells at different stages of differentiation, as well as noncancer cells from the tumour microenvironment. These cells exert specific functions further sustaining cancer progression, by maintaining a proinflammatory environment, by inducing activation of angiogenesis and by evading immune responses. The cellular heterogeneity implies more subtle forms of mutual interaction and communication among different cells and with the microenvironment, which may be the niche for future innovative anticancer therapeutic interventions.In this renewed view of cancer etiology, unexpected crosstalk is emerging between metabolic mediators/processes and pathological alterations.This special issue focuses on “Metabolism and cancer: old and new players” and intends to stimulate the discussion about new emerging targetable mediators of cancer metabolism and to evaluate possible noncanonical roles of old players in tumorigenesis as well as in cancer metabolism.Alternative cell death pathways and cell metabolism,” draws attention to the programmed form of necrosis, namely, necroptosis, and discusses the relevance of metabolic pathways involved in its modulation. The author suggests a possible interconnection between alterations of redox signaling pathways mediating necroptosis and mitochondrial impairment, which implies the latter as a crucial regulator of redox imbalance. The impairment of cancer cell viability by induction of cell death remains the final outcome of many anticancer strategies. Besides apoptosis, other modes of cell death have been defined and are attracting interest as alternative therapeutically exploitable approaches to impact apoptosis-resistant forms of cancer. The review by S. Fulda, “Targeting metabolism to induce cell death in cancer cells and cancer stem cells,” addresses the relationship between cancer cell metabolism and evasion from apoptosis by comparing the main features of cancer cells and cancer stem cells. The authors correlate the peculiar metabolic characteristics of these two types of cancer cells with their ability to evade apoptosis.An ideal anticancer therapy should be able to efficiently target cancer stem cells. This requires the discovery of markers selectively identifying malignant stem cells. The review by C. Pecqueur et al., “Functions of Bcl-xL at the interface between cell death and metabolism,” reviews new and intriguing properties of the Bcl-2 homolog Bcl-xL (B-cell lymphoma extra-large) linked to regulation of bioenergetic metabolism, which in turn controls important processes including mitochondrial ATP synthesis, Ca2+ flux, autophagy, mitosis, and protein acetylation.Over the last 10 years, noncanonical roles for many factors implicated in apoptosis were published. The family of B-cell lymphoma-2 (Bcl-2) proteins represents one of the most interesting examples, with several members exhibiting essentially pro- and apoptotic functions. The paper by J. Michels et al., “Mitochondrial complex I inhibitors and forced oxidative phosphorylation synergize in inducing cancer cell death,” shows that mitochondrial complex I inhibitors sensitize cancer cells to cell death under glucose depletion. Interestingly, combined treatments affecting glycolysis and leading to mitochondria impairment are ineffective on immortalized cells or cancer cells cultivated under high glucose conditions. Their results suggest that the forced switch from glycolysis to oxidative phosphorylation combined with the use of mitochondrial inhibitors may be used as alternative therapeutic approach to increase the sensitivity of cancer cells to death.Cancer cells rely on glycolysis rather than on oxidative phosphorylation to fulfill their energy needs. In conditions of glucose deprivation, however, cancer cells may reactivate mitochondrial bioenergetics as part of a prosurvival strategy. The study by R. Palorini et al., “PKM2, a central point of regulation in cancer metabolism,” gives an overview of our current knowledge about this important enzyme, discussing several of its potential contributions to tumorigenesis.Identification of specific metabolic intermediates as targets for future anticancer treatments may be at the basis for attempting a reversal of tumor metabolism to normal conditions, with the rationale of impairing cancer growth. Pyruvate kinase M2 (PKM2) catalyzing the final rate-limiting reaction of glycolysis currently attracts much interest because of its multiple emerging intracellular functions. The review by N. Wong et al., “Natural compounds as regulators of the cancer cell metabolism,” covers this aspect, focusing on natural compounds as untapped potential regulators of cancer cell metabolism. The authors discuss some of the most important pathways and factors implicated in altered cancer metabolism and give an overview of compounds extracted from natural sources potentially targeting these different aberrantly regulated metabolic key intermediates.A limiting factor in exploiting cancer metabolism for therapeutic purposes is the lack of availability of agents acting as specific modulators of cancer metabolism. The review by C. Cerella et al., “Role of insulin-like growth factor binding protein-3 in 1, 25-dihydroxyvitamin-D3-induced breast cancer cell apoptosis,” aims at identifying the mechanisms involved in 1, 25-dihydroxyvitamin D3-induced apoptosis in breast cancer cells. Their strategy suggests a role for insulin-like growth factor binding protein-3 (IGFBP-3) in vitamin D-induced apoptotic signaling and the involvement of impaired secretion of IGFBP-3 in acquired chemoresistance. The link between obesity, insulin resistance, and cancer is explored from different sides in three papers of this special issue, interestingly dealing all with hormonal forms of cancer. The paper by C. Brosseau et al., “C-terminal binding protein: a molecular link between metabolic imbalance and epigenetic regulation in breast cancer,” introduces the reader to the concept of metabolic transduction by stressing the link between shifts in carbohydrate metabolism and alterations in epigenetic regulatory mechanisms. In particular, these authors investigate the family of transcriptional repressors called C-terminal binding proteins (CtBPs), whose function is controlled by NAD+ levels, as an example of factors interacting and modulating the activity of different histone deacetylases (HDACs) depending on the metabolic status.The review by J. S. Byun and K. Gardner, “New players for advanced prostate cancer and the rationalisation of insulin-sensitising medication,” points to the roles played by metabolic disorders  in favoring anticancer treatment failure and consequent higher cancer-specific mortality. The authors discuss the promising use of insulin-sensitizing drugs as potential anticancer agents to be used in combinational therapies. Finally, the review by J. H. Gunter et al., “Although alterations in cellular metabolism are an old acquaintance in cancer etiology, we still need to do more before we can translate our knowledge into new and specific anticancer therapies. This final outcome will mostly depend on an in-depth identification of the mutual modulation between metabolism processes and factors that regulate proliferation and death/survival of cancer cells. With this special issue, we hope to offer new and relevant insights towards a critical reevaluation of this rapidly moving field.Claudia CerellaClaudia CerellaCarine MichielsCarine MichielsRoderick H. DashwoodRoderick H. DashwoodYoung-Joon SurhYoung-Joon SurhMarc DiederichMarc Diederich


Predators are a ubiquitous presence in most natural environments. Opportunities to contrast the behaviour of a species in the presence and absence of predators are thus rare. Here we report on the behaviour of howler monkey groups living under radically different conditions on two land-bridge islands in Lago Guri, Venezuela. One group of 6 adults inhabited a 190-ha island (Danto) where they were exposed to multiple potential predators. This group, the control, occupied a home range of 23 ha and contested access to food resources with neighbouring groups in typical fashion. The second group, containing 6 adults, was isolated on a remote, predator-free 0.6 ha islet (Iguana) offering limited food resources. Howlers living on the large island moved, fed and rested in a coherent group, frequently engaged in affiliative activities, rarely displayed agonistic behaviour and maintained intergroup spacing through howling. In contrast, the howlers on Iguana showed repulsion, as individuals spent most of their time spaced widely around the perimeter of the island. Iguana howlers rarely engaged in affiliative behaviour, often chased or fought with one another and were not observed to howl. These behaviors are interpreted as adjustments to the unrelenting deprivation associated with bottom-up limitation in a predator-free environment. Top-down and bottom-up regulated populations lie at the poles of a spectrum of possibilities. Under the top-down scenario, predators regulate prey numbers such that prey populations are maintained at densities below carrying capacity, and food does not limit numbers. Under bottom-up regulation, predation is absent and the population is free to increase until it reaches carrying capacity, at which point it becomes limited by its food supply 2 hydroelectric impoundment, Lago Guri, in the State of Bolivar, Venezuela Alouatta seniculus, hereafter simply howler), in two contrasting situations: 1) a ‘normal’ population on a large 190 ha island (Danto), and 2) a predator-free population living at ultrahigh density on a tiny 0.6 ha island (Iguana). The two howler populations became isolated in 1986 when water rising behind the Raul Leoní dam created hundreds of land-bridge islands ranging in size from <<1 ha to ca 750 ha The special circumstances created by the formation of a 4,300 km2, or more than 10 times that of howlers on Danto or howlers living in similar dry forest habitat on the Venezuelan mainland Although group sizes were similar on both the small and large islands, the circumstances of the groups were markedly different. Danto howlers occupied a normal-sized home range containing thousands of trees (minimum polygon of 23 ha), whereas the 0.6 ha available to Iguana howlers supported only 351 trees ≥10 cm dbh and many fewer tree species Panthera onca), puma (Puma concolor), ocelot , harpy eagle (Harpia harpyja), solitary eagle , boa constrictor (Constrictor constrictor), anaconda (Eunectes murinus) We observed groups of howlers on both islands in 1999 and 2000. Located <1 km from the mainland, Danto supported or was occasionally visited by several predators at least potentially capable of preying on howlers: jaguar . Both islands were covered in semi-evergreen tropical dry forest dominated by legumes and containing ca 50–70 species of trees per ha ad libitum.Howlers confined to tiny Lago Guri islets proved to be extremely shy and several years of persistent effort were required to habituate the Iguana group. Once habituation was adequate, we conducted observations using dawn-to-dark follows and continuous sampling of feeding and behaviour, following To assess the condition of howlers living at abnormally high densities, we captured and weighed 8 adult individuals  taken from 5 small (<1 ha) Lago Guri islands in August, 2001. Monkeys were captured using the method described in Statistical evaluations of categorical behaviours were conducted via Mann-Whitney U test and the analysis of weight differences between howlers captured on small Lago Guri islands and the Venezuelan mainland were conducted using ANOVA.Captured monkeys were examined and released into the wild within a few hours of capture. They were not caged or fed and proved virtually free of parasites, so no treatment was administered. After inspection and weighing, they were transported across 5–7 km of open water while recovering from anesthesia and released on the nearby mainland into the forested watershed protection zone of the Guri impoundment. They were handled gently at all times. Duke University Institutional Animal Care and Use Committee approved the capture protocol, including capture, handling, measuring and relocation, under #AO73-96-2. EDELCA, the hydroelectric company that managed the Guri impoundment granted permission to capture and release the animals.inter se, though dominant males are aggressive toward their counterparts in neighbouring groups inter se. Danto howlers shared overlapping home ranges with neighbouring groups (degree of overlap not quantified) and frequently contested possession of resource-bearing trees with loud howling matches. Agonistic behaviour was thus almost entirely restricted to between-group, rather than within-group, interactions.Howlers typically live in cohesive groups led by a dominant adult male. The members of such groups travel, feed and rest together and display little agonistic behaviour Confined to a home range less than 5% of the typical area for the species, and isolated from other conspecifics, Iguana howlers exhibited radically different behaviour . InsteadIn the absence of encounters with other groups, Iguana howlers did not howl, although the dominant male showed clear interest in the howling of other males on neighbouring islands by raising his head at the onset of a howling bout and orienting toward the source of the sound.Frequent conflicts over access to resource trees generated high levels of within-group conflict among Iguana howlers . Most agEvidence of dietary stress was obtained through the capture of 8 howlers  on 5 small islands . After w2 through 2002 when we last observed them. Such high densities are unknown on the nearby Venezuelan mainland where the same species normally occurs at densities of <100 per km2Howlers were trapped on the 0.6 ha island of Iguana in 1986 with the creation of Lago Guri and persisted at a density equivalent to 1000 per km2 suggests that the carrying capacity of the dry forest habitat is much greater than that suggested by the densities considered normal for the species in this habitat Iguana iguana) and leaf-cutter ants (Atta spp.) Persistence of Iguana howlers for 16 years (1986–2002) at a density equivalent to 1000 individuals/kmCrowding and food deprivation among Iguana howlers was associated with extraordinary differences in behavior in comparison to the Danto group that lived under more normal circumstances. Resource scarcity plausibly led to increased within-group competition for food resources among Iguana howlers Hierarchical access to food resources may lie behind the observation that the body weights of males from tiny islands similar to Danto were closer to those of males living on the mainland (88%) than was the case with females (76%). Iguana females also manifested a very low reproductive rate of 0.125 birth per female-year vs. 0.5 per female-year for Danto females Confinement to a tiny isolated island carries with it the absence of any threat of group takeovers by unrelated males and associated infanticide Howlers occupying small islands in Lago Guri live in a world regulated from the bottom up. Underweight, perpetually intimidated by higher-ranking individuals and obliged to subsist on non-preferred food resources, they live lives of social and caloric deprivation


Distal humeral periprosthetic fractures below intramedullary nail devices are complex and challenging to treat, in particular due to the osteopenic/porotic nature of bone found in these patients. Fixation is often difficult to satisfactorily achieve around the intramedullary device, whilst minimising soft tissue disruption. Descriptions of such cases in the current literature are very rare. We present the case of a midshaft humeral fracture treated with a locking compression plate that developed a nonunion, in a 60-year old female. This went on to successful union after exchange for an intramedullary humeral nail. Unfortunately, the patient developed a distal 1/5th humeral periprosthetic fracture, which was then successfully addressed with a single-contoured, extra-articular, distal humeral locking compression plate (Synthes) with unicortical locking screws and cerclage cables proximally around the distal nail tip region. An excellent postoperative range of motion was achieved. Fractures occurring below a humeral intramedullary nail are rare and present a unique challenge to management. Distal fixation is generally difficult due to osteopenia/porosis, and the presence of an intramedullary nail also poses difficulty in achieving reasonable proximal fixation. We present a case of a periprosthetic distal humeral fracture  that was successfully managed using a contoured locking plate. A 60-year-old right hand dominant occupational therapist presented to the emergency department with a left arm injury following a mechanical fall down a set of stairs. Her past medical history included controlled hypertension and mild asthma. She had a BMI of 36, was a nonsmoker, and consumed approximately 8–10 units of alcohol per week. A transverse, angulated, midshaft fracture of the left humerus  was confAt five months postoperative, radiographic review unfortunately confirmed an oligotrophic non-union with proximal screw breakage . This waRadiographs at 2 months following this revision demonstrated successful midshaft fracture union ; howeverThe elbow pain persisted despite removal of the distal locking screws and appropriate physiotherapy. There was no history of trauma. In particular, she experienced pain in the terminal 30° of extension. Plain radiographs demonstrated a transverse fracture of the distal 1/5th of the humerus propagating through the distal locking screw site, which, despite conservative treatment with bracing for 2 months, progressed to a hypertrophic non-union . This non-union was addressed via a posterior triceps split, debridement of the non-union site, application of DBX putty (Synthes), and fixation with an extra-articular distal humeral locking compression plate (Synthes). Proximal fixation was achieved with two unicortical locking screws supplemented with two tensioned 1.0 mm cables. This was uneventful, and progressive postoperative physiotherapy was initiated. At 5 months following this fixation, symptomatic and radiographic union was confirmed  with a fPeriprosthetic distal humeral fractures are uncommon and represent complex injuries to manage. There have been a number of reports in the literature on the management of distal humeral fractures occurring near the humeral component of either a shoulder arthroplasty or an elbow arthroplasty. Fewer still are descriptions of such fractures occurring in the distal humerus below an intramedullary nail. The region distal to the locking screws of an intramedullary humeral nail represents an area of increased stress resulting in an increased risk of fracture. Risk factors associated with periprosthetic distal humeral fractures include advancing age, female sex, osteoporosis, and rheumatoid arthritis . We are Shin et al.  describeSarraf et al. describe a distal humeral fracture in a 72-year-old man following intramedullary fixation of a midshaft humeral fracture . FracturCertainly, the development of locking plate technology has changed the way these and other complex periprosthetic fractures are managed, especially in osteoporotic bone . GreaterWhilst periprosthetic fractures of the distal humerus are uncommon, they present a unique challenge to management given the osteopenic/porotic nature of the bone. Our presented case highlights the importance of vigilant followup. More importantly, we demonstrate how these difficult fractures can be managed successfully with the use of a single-contoured locking plate with a long working length to prevent excessive rigidity and reduce strain on the proximal fixation provided by a combination of unicortical locking screws and cables around the region overlapped with the humeral nail. Soft tissue management is paramount to maintaining vascularity to the fracture region and maximising healing potential.


The world health organization has identified high blood pressure (BP) as one of the most important modifiable risk factors to reduce rapidly escalating burden of cardiovascular (CV) disease. Globally, 13.5% of total premature deaths annually (i.e. 7.6 million deaths) are directly attributed to hypertension.34Several guidelines in these guidelines are available to help physicians achieve better BP control.nd line agents. With this regard there have been two important publications recently.1213Observational studies and clinical trials have shown that commonly used antihypertensive agents exert variable BP- lowering response in ethnic populations. For example, compared to white Caucasians, the black African origin patients exhibit significantly poor BP lowering response to beta-blocker (B drug), ACE inhibitors or ARB’s (A drug), and much better response to CCB (C drug) and diuretics (D drug) when used as monotherapy –11. ThesNew analysis12These new studies have unequivocally shown that there are clinically important differences in BP-lowering response to both first- and second-line antihypertensive agents among hypertensive patients from different ethnic groups. These data illustrates the limitations in interpreting the results of data arising mainly from Caucasian populations to other ethnic groups, such as south-Asians and middle-Eastern origin patients. It also highlights urgent need of conducting clinical trials on patients from other ethnic groups. Pending these results, it is fair to suggest that current data on the use of antihypertensive agents suggest that one size do not fits all, and local  groups should modify these international guidelines for native consumption. Equally, it is important for the major guideline groups to promote the concept of heterogeneity in response to medications, and provide BP treatment algorithms which take into account patient specific conditions such as race, age, presence of obesity and other co-morbidities, whilst making a choice of first and second line agents for secondary prevention issues.


Trp53-mutated breast cancer, Brca1- and Brca2-associated hereditary breast cancer, and E-cadherin (Cdh1) mutated lobular breast cancer.Here we present the first paired-end sequencing of tumors from genetically engineered mouse models of cancer to determine how faithfully these models recapitulate the landscape of somatic rearrangements found in human tumors. These were models of Brca1- and Brca2-deficient mouse mammary tumors have a defect in the homologous recombination pathway, there is no apparent difference in the type or frequency of somatic rearrangements found in these cancers when compared to other mouse mammary cancers, and tumors from all genetic backgrounds showed evidence of microhomology-mediated repair and non-homologous end-joining processes. Importantly, mouse mammary tumors were found to carry fewer structural rearrangements than human mammary cancers and expressed in-frame fusion genes. Like the fusion genes found in human mammary tumors, these were not recurrent. One mouse tumor was found to contain an internal deletion of exons of the Lrp1b gene, which led to a smaller in-frame transcript. We found internal in-frame deletions in the human ortholog of this gene in a significant number (4.2%) of human cancer cell lines.We show that although Paired-end sequencing of mouse mammary tumors revealed that they display significant heterogeneity in their profiles of somatic rearrangement but, importantly, fewer rearrangements than cognate human mammary tumors, probably because these cancers have been induced by strong driver mutations engineered into the mouse genome. Both human and mouse mammary cancers carry expressed fusion genes and conserved homozygous deletions. Cancers form in humans as a result of the accumulation of mutations that co-operate together in subversion of growth control and the cell death signals that would normally result in apoptosis. Somatic mutations in cancer genomes can be classified as those that contribute to the evolution of the cancer, so-called 'driver mutations', and 'passenger mutations' that can be used to reveal the signature of the underlying mutagenic process, but do not contribute to tumorigenesis. Generally, passenger mutations are thought to substantially outnumber driver mutations, meaning that functional validation is generally important to distinguish between these types of mutations. This complexity has led to the development of genetically engineered mouse models (GEMMs) that aim to faithfully recreate features of human cancers and in so doing create a platform for assessing the causality of candidate cancer genes . Recentlc-Myc, EviI and Bcl11a/b are just a few genes discovered in this way .Reads that failed to align in the expected orientation or distance apart were further evaluated using the SSAHA algorithm to remove mapping errors in repetitive regions of the genome. In addition, during the PCR enrichment step, multiple PCR products derived from the same genomic template can occasionally be sequenced. To remove these, reads where both ends mapped to identical genomic locations (plus or minus a single nucleotide) were considered PCR duplicates, and only the read pair with the highest mapping quality retained. Further, erroneous mapping of reads originating from DNA present in sequence gaps in NCBI build m37 assembly were removed by excluding the highly repetitive regions within 1 Mb of a centromeric or telomeric sequence gap. Additional read pairs, where both ends mapped to within less than 500 bp of one another, but in the incorrect orientation, were excluded from analysis, unless support for a putative rearrangement was indicated by additional read pairs. The majority of these singleton read pairs are likely to be artifacts resulting from either intramolecular rearrangements generated during library amplification or mispriming of the sequencing oligonucleotide within the bridge amplified cluster. Finally, read pairs where both ends mapped to within 500 bp of a previously identified germline structural variant were removed from further analysis, as these are likely to represent the same germline allele.Generation of high-resolution copy number plots has been described previously ,12,33. BThe following criteria were used to determine which incorrectly mapping read pairs were evaluated by confirmatory PCR: 1, reads mapping ≥10 kb apart spanned by ≥2 read-independent read pairs ; 2, reads mapping ≥10 kb apart spanned by 1 read pair , with both ends mapping to within 100 kb of a change point in copy number identified by the segmentation algorithm; 3, reads mapping ≥600 bp apart spanned by ≥2 read-independent read pairs  with both ends mapping to within 100 kb of a change point in copy number identified by the segmentation algorithm; 4, selected read pairs mapping between 600 bp and 10 kb apart spanned by ≥2 independent read pairs . Primers were designed to span the possible breakpoint and to generate a maximum product size of 1 kb. PCR reactions were performed on tumor and normal genomic DNA for each set of primers at least twice, using the following thermocycling parameters: 95°C × 15 minutes  for 30 cycles, 72°C × 10 minutes. Products giving a band were sequenced by conventional Sanger capillary methods and compared to the reference sequence to identify breakpoints. Somatically acquired rearrangements were defined as those generating a reproducible band in the tumor DNA with no band in the normal (spleen) DNA following PCR amplification, together with unambiguously mapping sequence data suggesting a rearrangement. To support the somatic origin of the rearrangements identified in this study, we compared our calls to known structural variants [All breakpoints defined to the base-pair level were used in the analysis of breakpoint sequence context, excluding shards and overlapping regions. Analysis was performed on all breakpoints together, and also on subsets divided into deletions, tandem duplications, amplicons, other intrachromosomal events, and all interchromosomal events. We extracted 10 bp and 100 bp on either side of the breakpoint sites for analysis.RNA was extracted from mouse mammary tumor samples using Trizol  and reverse transcribed using random hexamers. Three combinations of two forward and two reverse PCR primers were designed to span the fusion breakpoints. Primer sequences are shown in Additional file Trp53 . The PCR reactions and capillary sequencing were performed in duplicates on tumor and normal DNA samples following standard protocols. The resulting traces were aligned against the mouse reference genome (NCBIM37) using the BLAST algorithm. We obtained 90.2% sequence coverage of Trp53 in PD3685a and 90.6% sequence coverage of this gene in PD3686a. Traces were manually inspected to identify potential somatic mutations. Primer sequences are provided in Additional file Primers were design to amplify all exons of LRP1B gene. We analyzed the PICNIC output for all cell lines flagged with homozygous deletions at the LRP1B locus. PICNIC assigns an absolute copy number score to SNP6 probes [We used the web-based analysis tool CONAN to determine which cell lines had a homozygous deletion in the 6 probes ,27. To daCGH: array comparative genomic hybridization; bp: base pair; GEMM: genetically engineered mouse model; HR: homologous recombination; MHMR: microhomology-mediated repair; NHEJ: non-homologous end-joining; PARP: poly(ADP-ribose) polymerase; PE-MPS: paired end massive parallel sequencing.IV, CK, PJS, LJM, HvG and ES performed experiments. LS, DG, PJC, and LFAW performed data analysis. MRS, JJ, PAF and DJA designed the research. CK and DJA wrote the paper.Trp53 (p53) in eight tumors sequenced as part of this studyA Southern blot hybridization showing the status of . The PCR primers shown were used to generate a Southern blot probe that was hybridized with size-fractioned genomic DNA. The 'wt' band represents the wild-type allele. The 'del' band represents the recombined Trp53 allele.Click here for fileAn overview of the sequencing and sequence read mapping metrics for the eight mouse mammary cancers sequenced as part of this study.Click here for fileA list of somatic structural rearrangements found in eight mouse mammary cancers. Each row represents a single somatically acquired rearrangement. The 'Sample' column represents the mammary cancer that was sequenced as part of this study in which the rearrangement was found. 'No. Reads' denotes the number of mapped reads spanning the rearrangement. 'Simplified Nomenclature' refers to the type of rearrangement. The 'Chr1' column represents the chromosome on which the rearrangement is resident. For intrachromosomal rearrangements the 'Chr2' column will denote the same chromosome as shown in the Chr1 column. For interchromosomal rearrangements Chr2 will carry a different chromosome. Position and strand represent the location of the rearrangement and the strand to which the sequence reads have mapped. The 'Size' column represents the distance between the mapped reads for an intrachromosomal rearrangement while the 'ReadName' column reports a single read that uniquely identifies the rearrangement.Click here for fileLRP1b locus in 102 sporadic human breast cancersCopy number analysis of the .Click here for filePrimers used for fusion gene validation.Click here for fileTrp53Primers used to sequence .Click here for file


Molecular MRI (mMRI) is a special implementation of Molecular Imaging for the non-invasive visualisation of biological processes at the cellular and molecular level. More specifically, mMRI comprises the contrast agent-mediated alteration of tissue relaxation times for the detection and localisation of molecular disease markers , cells  or therapeutic drugs . MRI yields topographical, anatomical maps; functional MRI (fMRI) provides rendering of physiologic functions and magnetic resonance spectroscopy (MRS) reveals the distribution patterns of some specific metabolites. mMRI provides an additional level of information at the molecular or cellular level, thus extending MRI further beyond the anatomical and physiological level. These advances brought by mMRI are mandatory for MRI to be competitive in the age of molecular medicine. mMRI is already today increasingly used for research purposes, e.g. to facilitate the examination of cell migration, angiogenesis, apoptosis or gene expression in living organisms. In medical diagnostics, mMRI will pave the way toward a significant improvement in early detection of disease, therapy planning or monitoring of outcome and will therefore bring significant improvement in the medical treatment for patients.15 per ml, i.e. the concentration is in the micromole range. Many targets, however, are expressed in even considerably lower concentrations. Therefore the most sensitive modalities, namely nuclear imaging (PET and SPECT) have always been at the forefront of Molecular Imaging, and many nuclear probes in clinical use today are already designed to detect molecular mechanisms . In recent years however, Molecular Imaging has commanded attention from beyond the field of nuclear medicine. Further imaging modalities to be considered for molecular imaging primarily include optical imaging, MRI and ultrasound.In general, Molecular Imaging demands high sensitivity equipment, capable of quantitative measurements to detect probes that interact with targets at the pico- or nanomolar level. The challenge to detect such sparse targets can be exemplified with cell surface receptors, a common target for molecular imaging. At high expression levels (bigger than 106 per cell) the receptor concentration is approx. 10 The above-mentioned concentration of molecular imaging targets in the micromolar range is challenging and requires sophisticated imaging strategies. Improvements in MRI design to reduce the lower detection limit are possible only to a certain extent; hence biophysical amplification mechanisms to enhance the signal from the label are necessary.Clinical MRI scanners offer a spatial resolution of 250 µm in-plane  or in T1-weighted images . For both classes, methods for signal amplification have been developed. In general, both take advantage of either very high relaxivity probes, background reduction (SNR optimisation) via activation of low-relaxivity probes by the targeted molecular marker (induced changes in relaxivity) or pronounced tissue accumulation. The latter is possible only with a very restricted number of highly expressed molecular marker (e.g. fibrin for thrombosis imaging).-1 [For T2-contrast agents, the most prominent labels are iron-oxide nanoparticles (Superparamagnetic Iron Oxide (SPIO), Very Small Paramagnetic Iron Oxide (VSPIO) or Ultrasmall Superparamagnetic Iron Oxide (USPIO)). These particles usually consist of a crystalline iron-oxide core, surrounded by polymer coating, often dextran, polyethyleneglycol or citrate. The advantage of these preparations is that each particle contains thousands of iron atoms resulting in very high T2 relaxivities of up to 200 (mMs)in vivo (cell tracking). Using this method, the spatial distribution of immuno-competent cells into tumours over time can be studied as well as the movement of stem cells, neuronal cells or blood stem cells in vivo [et al [Specific population of cells can be labeled with magnetic nanoparticles and followed  in vivo . A widelo [et al . Key conSimilar to PET-imaging, MRI can be used to image gene expression and to assess the efficiency of gene delivery. Viral or non-viral gene therapy schemes require targeting of the gene therapy vector to the tissue or cells of interest. Delivery of a therapeutic gene can be monitored by imaging of a reporter gene, which is introduced into the gene therapy vector and expressed together with the therapeutic gene . The repin vitro method for high throughput evaluation of biological specimens.A method named “magnetic relaxation switch” facilitates the reversible activation of MR probes . Target--1 range [in vivo binding of small sized contrast agents to serum proteins [Typical T1-contrast agents are small molecular weight compounds containing a single Lanthanide chelate as contrast producing element (e.g. Gadolinium-DTPA). The tissue concentration necessary to image with these T1-contrast agents on a molecular level is considerably higher than the required concentration of iron-oxide particles, it has to be in the order of mMols, since T1 relaxivity values are usually only in the in the 5-80 (mMs)proteins . MultilaSimilar to T2 contrast agents, T1-contrast probes can be activated by different mechanisms, thus increasing the relaxivity of the agent. This target-mediated increase of relaxivity leads to an increased contrast in the presence of the target. An enzyme-mediated polymerisation (e.g. bymyeloperoxidases up-regulated in vulnerable plaques) of paramagnetic substrates into oligomers represents one possible approach. In contrast to the small monomeric substrates these polymers have a larger hydrodynamic diameter and slower rotational rate of paramagnetic metal chelates. This results in a higher longitudinal relaxivity, and since the oligomeres are eliminated much slower than the monomers all signal will come after a certain time only from the oligomere, retained at the side of the target's presence . Anotherin vivo in all organs. It furthermore allows for the measurement of the pH or the temperature. Multiparametric mMRI might become feasible as CEST agents with different absorption frequencies of the exchanging protons can be designed. The sensitivity of CEST can be improved further by incorporating larger number of exchangeable protons into a (polymeric) CEST agent. This has been accomplished by LIPOCEST agents, liposomes containing a paramagnetic shift reagent for water protons in their aqueous inner cavity [Chemical-exchange saturation transfer (CEST) agents are another emerging class of negative MRI contrast agents, which facilitate an activatable contrast. The concept of CEST can be utilised to obtain highly amplified targeted agents. The principle is based on the fact that many molecules in the body exchange protons with bulk water through the selective RF irradiation of exchanging protons. After irradiating a millimolar pool of metabolite the energy is transferred to a nearby pool of water, producing a strong increase in enhancement by means of chemical exchange. With CEST and its newer development paraCEST specific metabolites can be detected. Using this method, the concentration of metabolites such as glucose can be evaluated non-invasively r cavity .Finally an “old friend” in MRI is attracting increasing attention in the course of Molecular Imaging: Fluorine-19. Xeno-labels, i.e. elements with low physiological concentration in situ but appropriate gyro-magnetic properties proved to provide superb traceability for mMRI.To date molecular imaging is increasingly used in laboratory studies. High resolution MRI is well suited to screen mouse models for tumours and other abnormalities and can be applied to non-invasively follow up new experimental treatment strategies. While dedicated animal systems are available, clinical scanners are also very capable of obtaining high-resolution images. To further speed up translational research, a small animal MR scanner based on a clinical console has been introduced just recently . This system comes with an adapted clinical user interface and supports therefore most of the features and sequences used on clinical scanners. Standardisation of the system ensures easy protocol transfer from and to clinical scanners. Applications being developed for humans on clinical scanners will be available on ClinScan. On the other hand, any application being developed for animals can be easily transferred to human diagnostics. In addition, adaptations for clinical scanner are underway for experimental molecular imaging. These include, but are not limited to, implementing dedicated small animal protocols, (quantitative) analysis tools or dedicated small animal RF coils. In conclusion, MRI is increasingly suited to carry molecular imaging from animal models into clinical practice.When will molecular imaging enter clinical medicine? Hot (hyper-intense) spots, which add information on the molecular mechanisms underlying a disease could guide radiologist and would be highly beneficial. Hyper-intense hot spots can be caused by highly specific accumulation of paramagnetic contrast agents. Most macromolecular Gadolinium-chelates are in an early pre-clinical research stages and the time needed to reach clinical applicability is considerable. Furthermore, toxicity concerns due to the prolonged retention of heavily Gadolinium-loaded molecules within the organism must be disproved. Superparamagnetic contrast agents can give easily detectable positive signal in combination with imaging techniques such as off resonance imaging.Superparamagnetic iron-oxide based contrast agents are easily detectable on T2-weighted sequences, even better so on T2*-weighted images due to susceptibility effects. Iron-oxide particles currently in clinical use can be utilised for cellular imaging of phagocyting cells, if combined with peptides, helping to traverse the membrane; this applicability can be even expanded to any other cell type. Iron-oxide particles can furthermore be functionalised with a wide variety of biological active molecules such as targeting moieties of peptides, antibody (derivatives), nucleic acids or aptamers to increase specific binding. Therefore a wide variety of imaging agents for mMRI is emerging and can facilitate steps towards clinical mMRI. Translational research with iron-oxide particles will take advantage of a growing basis of installed high field MRI scanners, since the lower detection limit decreases with increasing field strength.Besides being a powerful tool for the acquisition of molecular information, MRI is capable to add anatomical or functional information  to molecular images acquired with other modalities such as PET, SPECT or optical imaging. Combinations of CT and PET are increasingly used in clinical diagnostics. But there are some unique advantages for MR PET. Isocentric and simultaneous measurements are possible, no re-positioning of the patient, MRI can provide navigator technique and may give attenuation correction for PET, there is a better soft tissue contrast, and lower radiation exposure enables follow up studies. The ongoing development in imaging software and computing hardware facilitates co-registration and image fusion of physically separated units without delay. But repositioning of the patient and time interval between the scans makes fusion of separately obtained images difficult and inherently imprecise. Integrated devices would clearly be preferable even for economic reasons. Simon Cherry pioneered MR PET integration.Just recently the first fully integrated experimental set-up (based on semiconductor detectors) for MR PET has been presented by Siemens . Academiin vitro diagnostics (IVD) and knowledge driven healthcare , cost-intensive (molecular) treatment schemata (such as cell- or gene therapy) or therapies of chronic diseases. In such instances, a rational therapy selection based on comprehensive diagnostic data is a decisive factor for efficient and cost sensitive patient care. One of the main cost drivers in medicine is inappropriate treatment over a prolonged time. From a medical point of view, many arising molecular therapy concepts are based on individualised drugs. These treatments must be tailored to the individual biochemical set-up or disease stage of each respective patient with the support of diagnostic data. Equipped with these patient-specific data, a therapy regime is selected, taking into account the different molecular defects for each disease as well as the particular clinical history and condition of a patient.An example of how mMRI could contribute to this scope is radiation treatment planning by semi-automated lymph nodal cancer staging using a nanoparticle-enhanced lymphotropic mMRI. Preclinical data proves that mMRI demonstrate great promise for improving quality of diagnosis in general for oncologic- (tumour angiogenesis imaging), cardiovascular- (vulnerable plaque imaging) and neurological (Alzheimer’s plaque imaging) diseases.


Accumulating evidence suggests that the circadian clock is closely associated with metabolic regulation. However, whether an impaired circadian clock is a direct cause of metabolic dysregulation such as body weight gain is not clearly understood. In this study, we demonstrate that body weight gain in mice is not significantly changed by restricting feeding period to daytime or nighttime. The expression of peripheral circadian clock genes was altered by feeding period restriction, while the expression of light-regulated hypothalamic circadian clock genes was unaffected by either a normal chow diet (NCD) or a high-fat diet (HFD). In the liver, the expression pattern of circadian clock genes, including Bmal1, Clock, and Per2, was changed by different feeding period restrictions. Moreover, the expression of lipogenic genes, gluconeogenic genes, and fatty acid oxidation-related genes in the liver was also altered by feeding period restriction. Given that feeding period restriction does not affect body weight gain with a NCD or HFD, it is likely that the amount of food consumed might be a crucial factor in determining body weight. Collectively, these data suggest that feeding period restriction modulates the expression of peripheral circadian clock genes, which is uncoupled from light-sensitive hypothalamic circadian clock genes. Various physiological and behavioral oscillations, such as sleep-wake cycles, body temperature, blood pressure, and hormone secretion, are associated with the circadian clock The circadian clock exists in the hypothalamic suprachiasmatic nucleus (SCN) and in peripheral tissues such as the liver and fat In this study, we investigated whether feeding period restriction affects body weight gain in mice fed a NCD or HFD. We also analyzed the gene expression profiles of circadian clocks from hypothalamus (SCN) and peripheral tissue in mice with different feeding periods. Our data suggest that feeding period restriction does not influence body weight gain, whereas it differently regulates circadian oscillations in peripheral tissues but not in hypothalamus (SCN).The mice were maintained according to the guidelines of the Seoul National University Animal Experiment Ethics Committee. The protocol was approved by the committee on the Ethics of Animal Experiments of the Seoul National University (Permit Number: SNU-100316-4). Mice were euthanized if they met any early removal criteria  to minimize suffering. Four-week-old male C57BL/6N mice were obtained from SAMTAKO BIO KOREA Co., Ltd. They were housed in individual cages for pair-feeding in 12 hr light/12 hr dark cycles. After a minimum 1-week stabilization period, ad libitum group mice (5 weeks old) were exposed to food freely, and the nighttime-fed mice were pair-fed with NCD and 60% HFD  for 4 weeks to match the amount of food of the daytime group . Only thTotal RNA was isolated from liver and hypothalamus as described previously The levels of plasma cholesterol and triglycerides were measured using Infinity reagents . Plasma glucose levels were measured with a freestyle blood glucose meter .t test or ANOVA (Student-Newman-Keuls comparison test as post hoc). P<0.05 was interpreted as being statistically significant.The results are shown as means ± SD. All statistical analyses were performed by Student To address the question of whether body weight gain is affected by feeding behavior and the accompanying changes in peripheral circadian clock genes, we investigated the effects of feeding period restriction on body weight. Feeding period restrictions were divided into the following three groups: 1) ad libitum, in which mice were freely exposed to food; 2) restriction feeding in daytime (RF Day), in which mice could access food only in daytime; and 3) pair-feeding in nighttime (PF Night), in which mice were given the same amount of food as the RF Day group but only at night . BecauseInterestingly, we observed that the RF Day and PF Night groups displayed a similar pattern of body weight gain, regardless of the diet (NCD or HFD) . CompareWe next investigated the expression of circadian clock genes upon feeding period restriction. To examine the expression profiles of circadian clock genes, mice were sacrificed at ZT2 and ZT14, which may reflect different circadian clock gene expression patterns in a 12∶12-hour light/dark cycle. Total RNA was isolated from liver and hypothalamus, representing peripheral clock and central clock tissues, respectively, and analyzed by qRT-PCR. As shown in It is well known that feeding is an important factor for regulating circadian clock genes and hepatic lipid and glucose metabolism Because feeding period restriction altered the expression of a subset of metabolic genes in the liver without causing body weight gain, we examined serum metabolites that are relevant to the whole-body energy state in different feeding period restriction groups. Although it has been reported that serum metabolite levels vary throughout the feeding-fasting cycle Obesity is characterized by increased adipose tissue and lipid metabolism, which are caused by chronic excesses of energy intake over energy expenditure. Very recent reports have suggested that feeding period restriction may be an important factor influencing body weight gain and leading to obesity In the present work, we investigated the relationship between body weight gain and feeding period restriction by providing the same amount of food either during the day or during the night. We observed that feeding period restriction altered the expression patterns of peripheral circadian clock genes, probably via nutrition-sensitive hormones. However, the expression of central circadian clock genes in the hypothalamus was not modulated by feeding restriction in either NCD or HFD. These data imply that the central and peripheral circadian clocks are independently regulated, at least at the transcriptional level, upon feeding period restriction It has been reported that HFD challenge disrupts behavioral and physiological circadian rhythms. For instance, HFD leads to alterations in the periods of the locomotor activity and canonical circadian clock genes Disruption of circadian clock genes is triggered by various stimuli such as changes in the light/dark cycle and food consumption. Interference of the central circadian clock by light/dark cycle modulation affects body weight gain Here, we have shown that feeding period restriction selectively alters the expression patterns of peripheral circadian clock genes without inducing body weight gain. Concomitantly, feeding-restricted mice show altered metabolic gene expression levels and plasma profiles. Therefore, it is plausible to speculate that the central circadian clock might play a key role in regulating body weight by modulating energy intake and expenditure. Consistent with this idea, it appears that total calorie intake is a major factor in body weight. In contrast, peripheral circadian clock genes sensitively respond to metabolic alterations, likely because of changes in nutrients and hormones. The exact molecular mechanism by which the central circadian clock influences body weight gain remains to be elucidated. In conclusion, our data suggest that feeding period restriction selectively modulates peripheral circadian clocks and glucose and lipid metabolism without significantly changing body weight.Figure S1Feeding period restriction changes plasma metabolites.A: Serum glucose levels in AL, RF Day, and PF Night NCD-fed mice at ZT2 and ZT14. B: Serum TG levels in AL, RF Day, and PF Night NCD-fed mice at ZT2 and ZT14. C: Serum cholesterol levels in AL, RF Day, and PF Night NCD-fed mice at ZT2 and ZT14. Each bar represents mean ± SD of each group of mice (n = 3), *P<0.05, **P<0.01.(PDF)Click here for additional data file.Table S1Sequences of primers for real-time PCR.(PDF)Click here for additional data file.