Extracellular matrix formation after transplantation of human embryonic stem cell-derived cardiomyocytes.

Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) for cardiac regeneration is hampered by the formation of fibrotic tissue around the grafts, preventing electrophysiological coupling. Investigating this process, we found that: (1) beating hESC-CM in vitro are embedded in collagens, laminin and fibronectin, which they bind via appropriate integrins; (2) after transplantation into the mouse heart, hESC-CM continue to secrete collagen IV, XVIII and fibronectin; (3) integrin expression on hESC-CM largely matches the matrix type they encounter or secrete in vivo; (4) co-transplantation of hESC-derived endothelial cells and/or cardiac progenitors with hESC-CM results in the formation of functional capillaries; and (5) transplanted hESC-CM survive and mature in vivo for at least 24 weeks. These results form the basis of future developments aiming to reduce the adverse fibrotic reaction that currently complicates cell-based therapies for cardiac disease, and to provide an additional clue towards successful engraftment of cardiomyocytes by co-transplanting endothelial cells.

HER2 shedding and serum HER2 extracellular domain: Biology and clinical utility in breast cancer.

The transmembrane protein HER2 is over-expressed in approximately 15% of invasive breast cancers as a result of HER2 gene amplification. HER2 proteolytic cleavage (HER2 shedding) generates soluble truncated HER2 molecules that include only the extracellular domain and the concentration of which can be measured in the serum fraction of blood. HER2 shedding also generates a constitutively active truncated intracellular receptor of 95kDa (p95(HER2)). Another soluble truncated HER2 protein (Herstatin), which can also be found in serum, is the product of an alternatively spliced HER2 transcript. Recent preclinical findings may provide crucial insights into the biological and clinical relevance of increased sHER2 concentrations for the outcome of HER2-positive breast cancer and sensitivity to trastuzumab and lapatinib treatment. We present here the most recent findings about the role and biology of sHER2 based on data obtained using a standardized test, which has been cleared by FDA in 2000, for measuring sHER2. This test includes quality control assessments and has been already widely used to evaluate the clinical utility of sHER2 as a biomarker in breast cancer. We will describe in detail data concerning the assessment of sHER2 as a surrogate maker to optimize the evaluation of the HER2 status of a primary tumor and as a prognosis and predictive marker of response to therapies, both in early and metastatic breast cancer.

Novel Agents Targeting the IGF-1R/PI3K Pathway Impair Cell Proliferation and Survival in Subsets of Medulloblastoma and Neuroblastoma.

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

A sensitized RNA interference screen identifies a novel role for the PI3K p110γ isoform in medulloblastoma cell proliferation and chemoresistance.

Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. We were interested in gaining further insight into the potential of targeting the human kinome as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. A library of small interfering RNA (siRNA) was used to downregulate the known human protein and lipid kinases in medulloblastoma cell lines. The analysis of cell proliferation, in the presence or absence of a low dose of cisplatin after siRNA transfection, identified new protein and lipid kinases involved in medulloblastoma chemoresistance. PLK1 (polo-like kinase 1) was identified as a kinase involved in proliferation in medulloblastoma cell lines. Moreover, a set of 6 genes comprising ATR, LYK5, MPP2, PIK3CG, PIK4CA, and WNK4 were identified as contributing to both cell proliferation and resistance to cisplatin treatment in medulloblastoma cells. An analysis of the expression of the 6 target genes in primary medulloblastoma tumor samples and cell lines revealed overexpression of LYK5 and PIK3CG. The results of the siRNA screen were validated by target inhibition with specific pharmacological inhibitors. A pharmacological inhibitor of p110γ (encoded by PIK3CG) impaired cell proliferation in medulloblastoma cell lines and sensitized the cells to cisplatin treatment. Together, our data show that the p110γ phosphoinositide 3-kinase isoform is a novel target for combinatorial therapies in medulloblastoma.

The Swiss registry for pulmonary arterial hypertension: the paediatric experience.

Pulmonary arterial hypertension is a rare disease with a poor prognosis. Epidemiological data are scarce, particularly in the paediatric population. A registry was recently developed in order to collect epidemiological data on patients with pulmonary arterial hypertension (PAH) in Switzerland. This is the first description of the paediatric data. Paediatric patients aged 0-18 years with the diagnosis of PAH were enrolled in the registry from 1999 to 2005 with informed consent from their parents. Patient characteristics, PAH aetiology, functional capacity, exercise capacity, treatments and outcome were among the most important data collected. A total of 23 patients (12 male, 11 female) have been thus far included in the registry. Median age at time of diagnosis was 3 years (range 1 month-18 years) and median follow-up was 3.47 years (range 1 day-12.6 years). PAH aetiologies are diagnosed as idiopathic in 8/23 patients (34.8%) and associated with congenital heart diseases in 12/23 (52.2%) or with pulmonary diseases in 3/23 patients (13.0%). Death occurred in 1 patient before treatment was initiated. Single treatments include medications with a calcium channel blocker in 2/23 patients, with bosentan in 10/23, and with inhaled iloprost in 1/23. Combined therapies include bosentan and inhaled iloprost in 7/23 patients, bosentan and sildenafil in 2/23 patients, and bosentan, sildenafil and inhaled iloprost in 2/23 patients. Additional oral anticoagulation is given to 14/23 patients and 8/23 patients are on oxygen therapy. NYHA class at baseline visit was obtained in 22/23 patients (4 NYHA 2, 17 NYHA 3 and 1 NYHA 4). Changes in NYHA class were observed over a 2-year period in 3/22 patients who improved from NYHA 3 to NYHA 2. Initial improvement of 6-minute walk distance was observed in 6/13 patients with a sustained improvement in 4. These preliminary results provide information on the epidemiology of PAH in children in Switzerland and demonstrate that most paediatric patients show stabilisation of the disease under new treatments. This underscores the utility of registries for rare diseases in providing crucial information in the era of new therapies. It may also help to improve the future medical approach.

Diagnosis of muscular dystrophies at the nanometer scale

The diagnosis of muscular dystrophies or the assessment of the functional benefit of gene or cell therapies can be difficult, especially for poorly accessible muscles, and it often lacks a singlefiber resolution. In the present study, we evaluated whether muscle diseases can be diagnosed from small biopsies using atomic force microscopy (AFM). AFM was shown to provide a sensitive and quantitative description of the resistance of normal and dystrophic myofibers within live muscle tissues explanted from Duchenne mdx mice. The rescue of dystrophin expression by gene therapy approaches led to the functional recovery of treated dystrophic muscle fibers, as probed using AFM and by in situ wholemuscle strength measurements. Comparison of muscles treated with viral or non-viral vectors indicated that the efficacy of the gene transfer approaches could be distinguished with a single myofiber resolution. This indicated full correction of the resistance to deformation in nearly all of the muscle fibers treated with an adeno-associated viral vector that mediates exon-skipping on the dystrophin mRNA. Having shown that AFM can provide a quantitative assessment of the expression of muscle proteins and of the muscular function in animal models, we assessed myofiber resistance in the context of human muscular dystrophies and myopathies. Thus, various forms of human Becker syndrome can also be detected using AFM in blind studies of small frozen biopsies from human patients. Interestingly, it also allowed the detection of anomalies in a fraction of the muscle fibers from patients showing a muscle weakness that could not be attributed to a known molecular or genetic defect. Overall, we conclude that AFM may provide a useful method to complement current diagnosis tools of known and unknown muscular diseases, in research and in a clinical context.

Invasive lobular breast cancer and its variants: How special are they for systemic therapy decisions?

The WHO classification of breast tumors distinguishes, besides invasive breast cancer 'of no special type' (former invasive ductal carcinoma, representing 60-70% of all breast cancers), 30 special types, of which invasive lobular carcinoma (ILC) is the most common (5-15%). We review the literature on (i) the specificity and heterogeneity of ILC biology as documented by various analytical techniques, including the results of molecular testing for risk of recurrence; (ii) the impact of lobular histology on prediction of prognosis and effect of systemic therapies in patients. Though it is generally admitted that ILC has a better prognosis than IDC, is endocrine responsive, and responds poorly to chemotherapy, currently available data do not unanimously support these assumptions. This review demonstrates some lack of specific data and a need for improving clinical research design to allow oncologists to make informed systemic therapy decisions in patients with ILC. Importantly, future studies should compare various endpoints in ILC breast cancer patients among the group of hormonosensitive breast cancer.

Opinions on tobacco control policies in Lausanne, Switzerland, 2003-2006.

OBJECTIVE: To assess the determinants of opinions regarding tobacco control policies in the Swiss general population. METHODS: Cross-sectional study conducted between 2003 and 2006 on a random sample of adult residents of Lausanne, Switzerland, aged 35-75 years (2601 women and 2398 men). Nine questions on smoking policies were applied. RESULTS: Ninety-five percent of responders supported policies that would help smokers to quit, 92% no selling of tobacco to subjects aged less than 16 years, 87% a smoking ban in public places and 86% a national campaign against smoking. A further 77% supported a total ban on tobacco advertising, 74% the reimbursement of nicotine replacement therapies and 70% an increase in the price of cigarettes. A lower support was found for two non-evidence-based interventions total ban of tobacco sales (35%) and promotion of light cigarettes (22%). Never smokers, women, physically active subjects, teetotallers and subjects with lower educational level were more likely to favour stronger measures while no differences were found between age groups. Reimbursement of nicotine replacement therapies was favoured more by current smokers and inactive subjects. CONCLUSION: The vast majority of responders supported the recommended tobacco control policies. Opinions regarding specific interventions vary according to the policy and subjects' characteristics.

Androgen dependence of hirsutism, acne, and alopecia in women: retrospective analysis of 228 patients investigated for hyperandrogenism.

Hirsutism, acne, alopecia, and oligo-amenorrhea are clinical expressions of hyperandrogenism, one of the most frequent endocrine disorders in women of reproductive age. Women referred to our endocrine clinics for skin symptoms of hyperandrogenism underwent a laboratory workup to evaluate hormone measurements and received antiandrogen therapy. We retrospectively analyzed the outcome of 228 consecutive patients investigated over 6 years.Patients with hirsutism had higher levels of androstenedione, dehydroepiandrosterone sulfate (DHEAS), and salivary testosterone; lower levels of sex hormone-binding globulin (SHBG); and a higher prevalence of oligo-amenorrhea than patients with alopecia, while patients with acne showed intermediate values. Hirsutism score correlated positively with androstenedione, DHEAS, and salivary testosterone, and correlated negatively with SHBG; salivary testosterone showed the highest correlation coefficient. Total testosterone was not significantly different among patients with hirsutism, alopecia, or acne, and did not significantly correlate with hirsutism score. Hirsutism and oligo-amenorrhea were the most sensitive symptoms of hyperandrogenism, and no androgenic parameter alone allowed us to identify all cases of hyperandrogenism.Patients of central European origin sought consultation with milder hirsutism scores than patients of southern European origin. There was, however, no difference in the clinical-biological correlation between these groups, arguing against differences in skin sensitivity to androgens.Polycystic ovary syndrome, defined as hyperandrogenism (hirsutism or elevated androgens) and oligo-amenorrhea, was diagnosed in 63 patients (27.6%), an underestimate compared with other reports that include systematic ovarian ultrasound studies. Neither pelvic ultrasound, used in a limited number of cases, nor the luteinizing hormone/follicle-stimulating hormone ratio helped to distinguish patients with polycystic ovary syndrome from the other diagnostic groups. These included hyperandrogenism (hirsutism or elevated androgens) and eumenorrhea (101 patients; 44.3%); normal androgens (acne or alopecia and eumenorrhea) (51 patients; 22.4%); isolated low SHBG (7 patients; 3.1%); nonclassical congenital adrenal hyperplasia (4 patients; 1.8% of total, 4.9% of patients undergoing cosyntropin stimulation tests); and ovarian tumor (2 patients; 0.9%).Ethinylestradiol and high-dose cyproterone acetate treatment lowered the hirsutism score to 53.5% of baseline at 1 year, and was also effective in treating acne and alopecia. The clinical benefit is ascribed to the peripheral antiandrogenic effect of cyproterone acetate as well as the hormone-suppressive effect of this combination. Salivary testosterone showed the most marked proportional decrease of all the androgens under treatment. Cost-effectiveness and tolerance of ethinylestradiol and high-dose cyproterone acetate compared well with other antiandrogenic drug therapies for hirsutism. The less potent therapy with spironolactone only, a peripheral antiandrogen without hormone-suppressive effect, was effective in treating isolated alopecia in patients with normal androgens.

Importance clinique du caryotype en hématologie. [Clinical importance of karyotype in hematology.]

Cytogenic analysis of leukemic cells has proven to be a mandatory part of the diagnosis of malignant hemopathies. Recurring clonal cytogenetic abnormalities may be divided into those exclusively associated with myeloid disorders, those uniquely observed in lymphoid diseases, and those detected in both myeloid and lymphoid hemopathies. Several of the common defects are characteristic of specific FAB types or subtypes and are associated with specific clinico pathologic syndromes and clinical complications. Cytogenetic abnormalities have served to define relatively homogeneous subsets of malignant hemopathies which are not evident from morphological and other available markers. Cytogenetic findings have been demonstrated to be powerful indicators in predicting clinical course and outcome in patients and in guiding their management. Given the significant progress made in the treatment of malignant hemopathies, it is very important to identify parameters which may be used to predict whether patients will respond favorably to standard therapies or if they are unlikely to do so and require alternative strategies, such as bone marrow transplantation. Cytogenetic studies have also provided important insights into the understanding of malignant transformation processes. In a number of recurring chromosome translocations characteristic of leukemias and lymphomas the genes that are located at the breakpoints have been identified. Molecular analysis has revealed that alteration in expression of these genes or in the properties of the encoded proteins resulting from the rearrangements plays an integral part in malignant transformation. Studies of clonality have suggested that several chromosome abnormalities may arise in pluripotent hemopoietic stem cells, whereas others may originate in cells of more restricted lineage. The author focuses first on the implications of the karyotype in the diagnosis and the prognosis of myeloproliferative syndromes, acute leukemias and myelodysplastic syndromes, then on the interest of describing new clinical-cytogenetic associations. Finally, some of the recent results obtained in a cytogenetic study of myelodysplastic syndromes are discussed.

Survival response in the skin

Apoptosis is defined as a programmed cell death process operating in multicellular organisms in order to maintain proper homeostasis of tissues. Caspases are among the best characterized proteases to execute apoptosis although lately many studies have associated them with non-apoptotic functions. In the laboratory an antiapoptotic pathway relying on caspase-3 activation and RasGAP has been described in vitro. RasGAP bears two conserved caspase-3 cleavage sites. Under low stress conditions, RasGAP is first cleaved by low caspase-3 activity generating an N terminal fragment (fragment N) that induces a potent anti-apoptotic response mediated by the Ras/PI3K/Akt pathway. High levels of active caspase-3, associated with increased stress conditions, induce further cleavage of fragment N abrogating this anti-apoptotic response. In the present work I studied the functionality of fragment N-mediated protection in physiological conditions as well as the mechanism by which fragment N induces an anti-apoptotic response, with a focus on survivin, an inhibitor of apoptosis. During my work in the laboratory I found that mice lacking caspase-3 or unable to cleave RasGAP (KI mice) are deficient in Akt activation and more sensitive to apoptosis than wild-type mice in response to stress. This higher sensitivity to stress led to augmented tissue damage, highlighting the importance of this pathway in protection against low stress. In parallel I focused on the study of survivin expression in the skin in response to UV-B light and I found that survivin is induced in the cytoplasm of keratinocytes in response to stress where it may fulfill a cyto-protective role. However fragment N had no effect on survivin expression. In addition, cytoplasmic survivin was increased in keratinocytes exposed to UV-B light, whether RasGAP is cleaved (WT mice) or not (KI mice), indicating that survivin is not involved in fragment N mediated protection. Altogether these data indicate that fragment N is pivotal for cell protection against pathophysiologic damage and can encourage the development of therapies aimed to strengthen the resistance of cells against aggressive treatments. Importantly, this finding contributes to the characterization of how caspase-3 can be activated without inducing cell death, although further studies need to be conducted in order to completely characterize this pro-survival molecular mechanism.

Development, optimization, and validation of novel anti-TEM1/CD248 affinity agent for optical imaging in cancer.

Tumor Endothelial Marker-1 (TEM1/CD248) is a tumor vascular marker with high therapeutic and diagnostic potentials. Immuno-imaging with TEM1-specific antibodies can help to detect cancerous lesions, monitor tumor responses, and select patients that are most likely to benefit from TEM1-targeted therapies. In particular, near infrared(NIR) optical imaging with biomarker-specific antibodies can provide real-time, tomographic information without exposing the subjects to radioactivity. To maximize the theranostic potential of TEM1, we developed a panel of all human, multivalent Fc-fusion proteins based on a previously identified single chain antibody (scFv78) that recognizes both human and mouse TEM1. By characterizing avidity, stability, and pharmacokinectics, we identified one fusion protein, 78Fc, with desirable characteristics for immuno-imaging applications. The biodistribution of radiolabeled 78Fc showed that this antibody had minimal binding to normal organs, which have low expression of TEM1. Next, we developed a 78Fc-based tracer and tested its performance in different TEM1-expressing mouse models. The NIR imaging and tomography results suggest that the 78Fc-NIR tracer performs well in distinguishing mouse- or human-TEM1 expressing tumor grafts from normal organs and control grafts in vivo. From these results we conclude that further development and optimization of 78Fc as a TEM1-targeted imaging agent for use in clinical settings is warranted.

Defining the critical hurdles in cancer immunotherapy.

Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.

Prognostic effect of epithelial cell adhesion molecule overexpression in untreated node-negative breast cancer.

PURPOSE: Epithelial cell adhesion molecule (Ep-CAM) recently received increased attention not only as a prognostic factor in breast cancer but also as a potential target for immunotherapy. We examined Ep-CAM expression in 402 consecutive node-negative breast cancer patients with long-term follow-up not treated in the adjuvant setting. EXPERIMENTAL DESIGN: Ep-CAM expression was evaluated by immunostaining. Its prognostic effect was estimated relative to overexpression/amplification of HER-2, histologic grade, tumor size, age, and hormone receptor expression. RESULTS: Ep-CAM status was positive in 106 (26.4%) patients. In multivariate analysis, Ep-CAM status was associated with disease-free survival independent of age, pT stage, histologic grade, estrogen receptor (ER), progesterone receptor (PR), as well as HER2 status (P = 0.028; hazard ratio, 1.60; 95% confidence interval, 1.05-2.44). Recently, so-called triple-negative (HER-2, ER, and PR) breast cancer has received increased attention. We noticed a similar association of Ep-CAM with disease-free survival in the triple-negative group as for the entire cohort. CONCLUSION: In this study of untreated breast cancer patients, Ep-CAM overexpression was associated with poor survival in the entire cohort and in the subgroup of triple-negative breast cancer. This suggests that Ep-CAM may be a well-suited target for specific therapies particularly in HER-2-, ER-, and PR-negative tumors.