Combination of trastuzumab and letrozole after resistance to sequential trastuzumab and aromatase inhibitor monotherapies in patients with estrogen receptor-positive, HER-2-positive advanced breast cancer: a proof-of-concept trial (SAKK 23/03).

A sequential treatment design was chosen in this trial to ensure complete resistance to single-agent non-steroidal aromatase inhibitor (AI) and trastuzumab both given as monotherapy before receiving the combination of a non-steroidal AI and trastuzumab. Key eligibility criteria included postmenopausal patients with advanced, measurable, human epidermal growth factor receptor-2 (HER-2)-positive disease (assessed by FISH, ratio (≥2)), hormone receptor (HR)-positive disease, and progression on prior treatment with a non-steroidal AI, e.g. letrozole or anastrozole, either in the adjuvant or in the advanced setting. Patients received standard dose trastuzumab monotherapy in step 1 and upon disease progression continued trastuzumab in combination with letrozole in step 2. The primary endpoint was clinical benefit rate (CBR) in step 2. Totally, 13 patients were enrolled. In step 1, six patients (46%) achieved CBR. Median time to progression (TTP) was 161 days (95% confidence interval (CI): 82-281). In step 2, CBR was observed in eight out of the 11 evaluable patients (73%), including one patient with partial response. Median TTP for all the 11 patients was 188 days (95% CI: 77-not reached). Results of this proof-of-concept trial suggest that complete resistance to both AI and trastuzumab can be overcome in a proportion of patients by combined treatment of AI and trastuzumab, as all patients served as their own control. Our results appear promising for a new treatment strategy that offers a chemotherapy-free option for at least a subset of patients with HR-positive, HER-2-positive breast cancer over a clinically relevant time period.

How to confirm C.E.R.A. doping in athletes' blood?

C.E.R.A. (Continuous Erythropoietin Receptor Activator) is a new third-generation erythropoiesis-stimulating agent that has recently been linked with abuse in endurance sports. The anti-doping community rapidly reacted by releasing a high-throughput screening ELISA allowing the detection of C.E.R.A. doping in athletes' blood. In order to return adverse analytical findings, anti-doping laboratories, however, need, as far as possible, to confirm the presence of the drug in athletes' samples through orthogonal methods. This article focuses on the comparison of 2 proposed confirmation assays based on gel electrophoresis that were coupled with a new sample immunopurification method. IEF, the classical method used to target erythropoietin (EPO) and its recombinant analogues in athletes' samples, and SARKOSYL-PAGE were applied to the plasma samples of subjects having received a single injection of C.E.R.A. It was demonstrated that SARKOSYL-PAGE was at least 6 times more sensitive than IEF, with comparable specificity. A longer detection window coupled with easier interpretation criteria led us to recommend the use of SARKOSYL-PAGE to confirm C.E.R.A. presence in athletes' blood.

Clinical and genetic findings in a large cohort of patients with ryanodine receptor 1 gene-associated myopathies.

Ryanodine receptor 1 (RYR1) mutations are a common cause of congenital myopathies associated with both dominant and recessive inheritance. Histopathological findings frequently feature central cores or multi-minicores, more rarely, type 1 predominance/uniformity, fiber-type disproportion, increased internal nucleation, and fatty and connective tissue. We describe 71 families, 35 associated with dominant RYR1 mutations and 36 with recessive inheritance. Five of the dominant mutations and 35 of the 55 recessive mutations have not been previously reported. Dominant mutations, typically missense, were frequently located in recognized mutational hotspot regions, while recessive mutations were distributed throughout the entire coding sequence. Recessive mutations included nonsense and splice mutations expected to result in reduced RyR1 protein. There was wide clinical variability. As a group, dominant mutations were associated with milder phenotypes; patients with recessive inheritance had earlier onset, more weakness, and functional limitations. Extraocular and bulbar muscle involvement was almost exclusively observed in the recessive group. In conclusion, our study reports a large number of novel RYR1 mutations and indicates that recessive variants are at least as frequent as the dominant ones. Assigning pathogenicity to novel mutations is often difficult, and interpretation of genetic results in the context of clinical, histological, and muscle magnetic resonance imaging findings is essential.

Effects of the peroxisomal proliferator-activated receptor-gamma agonist pioglitazone on renal and hormonal responses to salt in healthy men.

Glitazones are used in the treatment of type 2 diabetes as efficient insulin sensitizers. They can, however, induce peripheral edema through an unknown mechanism in up to 18% of cases. In this double-blind, randomized, placebo-controlled, four-way, cross-over study, we examined the effects of a 6-wk administration of pioglitazone (45 mg daily) or placebo on the blood pressure, hormonal, and renal hemodynamic and tubular responses to a low (LS) and a high (HS) sodium diet in healthy volunteers. Pioglitazone had no effect on the systemic and renal hemodynamic responses to salt, except for an increase in daytime heart rate. Urinary sodium excretion and lithium clearance were lower with pioglitazone, particularly with the LS diet (P < 0.05), suggesting increased sodium reabsorption at the proximal tubule. Pioglitazone significantly increased plasma renin activity with the LS (P = 0.02) and HS (P = 0.03) diets. Similar trends were observed with aldosterone. Atrial natriuretic levels did not change with pioglitazone. Body weight increased with pioglitazone in most subjects. Pioglitazone stimulates plasma renin activity and favors sodium retention and weight gain in healthy volunteers. These effects could contribute to the development of edema in some subjects treated with glitazones.

Differential effects of selective HDAC inhibitors on macrophage inflammatory responses to the Toll-like receptor 4 agonist LPS.

Broad-spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti- and proinflammatory effects on macrophages in vitro. We report here that several broad-spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS-induced mRNA expression of the proinflammatory mediators Edn-1, Ccl-7/MCP-3, and Il-12p40 but amplified the expression of the proatherogenic factors Cox-2 and Pai-1/serpine1 in primary mouse BMM. Similar effects were also apparent in LPS-stimulated TEPM and HMDM. The pro- and anti-inflammatory effects of TSA were separable over a concentration range, implying that individual HDACs have differential effects on macrophage inflammatory responses. The HDAC1-selective inhibitor, MS-275, retained proinflammatory effects (amplification of LPS-induced expression of Cox-2 and Pai-1 in BMM) but suppressed only some inflammatory responses. In contrast, 17a (a reportedly HDAC6-selective inhibitor) retained anti-inflammatory but not proinflammatory properties. Despite this, HDAC6(-/-) macrophages showed normal LPS-induced expression of HDAC-dependent inflammatory genes, arguing that the anti-inflammatory effects of 17a are not a result of inhibition of HDAC6 alone. Thus, 17a provides a tool to identify individual HDACs with proinflammatory properties.

Coactivated platelet-derived growth factor receptor {alpha} and epidermal growth factor receptor are potential therapeutic targets in intimal sarcoma.

Intimal sarcoma (IS) is a rare, malignant, and aggressive tumor that shows a relentless course with a concomitant low survival rate and for which no effective treatment is available. In this study, 21 cases of large arterial blood vessel IS were analyzed by immunohistochemistry and fluorescence in situ hybridization and selectively by karyotyping, array comparative genomic hybridization, sequencing, phospho-kinase antibody arrays, and Western immunoblotting in search for novel diagnostic markers and potential molecular therapeutic targets. Ex vivo immunoassays were applied to test the sensitivity of IS primary tumor cells to the receptor tyrosine kinase (RTK) inhibitors imatinib and dasatinib. We showed that amplification of platelet-derived growth factor receptor α (PDGFRA) is a common finding in IS, which should be considered as a molecular hallmark of this entity. This amplification is consistently associated with PDGFRA activation. Furthermore, the tumors reveal persistent activation of the epidermal growth factor receptor (EGFR), concurrent to PDGFRA activation. Activated PDGFRA and EGFR frequently coexist with amplification and overexpression of the MDM2 oncogene. Ex vivo immunoassays on primary IS cells from one case showed the potency of dasatinib to inhibit PDGFRA and downstream signaling pathways. Our findings provide a rationale for investigating therapies that target PDGFRA, EGFR, or MDM2 in IS. Given the clonal heterogeneity of this tumor type and the potential cross-talk between the PDGFRA and EGFR signaling pathways, targeting multiple RTKs and aberrant downstream effectors might be required to improve the therapeutic outcome for patients with this disease.

Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells.

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

Peroxisome proliferator-activated receptor-alpha-null mice have increased white adipose tissue glucose utilization, GLUT4, and fat mass: Role in liver and brain.

Activation of the peroxisome proliferator-activated receptor (PPAR)-alpha increases lipid catabolism and lowers the concentration of circulating lipid, but its role in the control of glucose metabolism is not as clearly established. Here we compared PPARalpha knockout mice with wild type and confirmed that the former developed hypoglycemia during fasting. This was associated with only a slight increase in insulin sensitivity but a dramatic increase in whole-body and adipose tissue glucose use rates in the fasting state. The white sc and visceral fat depots were larger due to an increase in the size and number of adipocytes, and their level of GLUT4 expression was higher and no longer regulated by the fed-to-fast transition. To evaluate whether these adipocyte deregulations were secondary to the absence of PPARalpha from liver, we reexpresssed this transcription factor in the liver of knockout mice using recombinant adenoviruses. Whereas more than 90% of the hepatocytes were infected and PPARalpha expression was restored to normal levels, the whole-body glucose use rate remained elevated. Next, to evaluate whether brain PPARalpha could affect glucose homeostasis, we activated brain PPARalpha in wild-type mice by infusing WY14643 into the lateral ventricle and showed that whole-body glucose use was reduced. Hence, our data show that PPARalpha is involved in the regulation of glucose homeostasis, insulin sensitivity, fat accumulation, and adipose tissue glucose use by a mechanism that does not require PPARalpha expression in the liver. By contrast, activation of PPARalpha in the brain stimulates peripheral glucose use. This suggests that the alteration in adipocyte glucose metabolism in the knockout mice may result from the absence of PPARalpha in the brain.

TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.

Lipoxin A4 Is A Novel Estrogen Receptor Agonist

Lipoxins (LXs), are endogenously produced eicosanoids, which possess potent antiinflammatory and proresolution bioactivities. The role of LXs in the endometrium is unknown. Our initial observations showed LXA4 enhanced estrogen receptor (ER)-mediated transcriptional activation in Ishikawa endometrial epithelial cells. Furthermore, we demonstrated that LXA4 possesses robust estrogenic activity through its capacity to alter cellular proliferation as well as the expression of estrogen-regulated genes implicated in cancer development. Interestingly, LXA4 also demonstrated antiestrogenic potential in that it attenuated E2-mediated cellular proliferation, consistent with the effects of a partial ER agonist. Subsequent studies revealed that these actions of LXA4 were directly mediated by ERa and appear to closely mimic those of the potent estrogen, 17b-Estradiol (E2). Using competitive radioligand binding assays, we confirmed that this lipid binds ER. We additionally demonstrated this estrogenic activity of LXA4 in mouse uterus in vivo using a uterotrophic assay and the expression of E2- dependent genes as readouts. Taken together our results establish a dual capacity of LXA4 to modulate estrogenic activity in the endometrium. These findings highlight a previously unappreciated paradigm in LXA4-mediated activities and reveal novel immunoendocrine crosstalk mechanisms. Disclosure of interest: None declared.

Clinical and hormonal effects of the new angiotensin II receptor antagonist LRB081.

The renin-angiotensin system is a major contributor to the pathophysiology of cardiovascular diseases such as congestive heart failure and hypertension. Antagonizing angiotensin (Ang) II at the receptor site may produce fewer side effects than inhibition of the promiscuous converting enzyme. The present study was designed to assess in healthy human subjects the effect of LRB081, a new orally active AT1-receptor antagonist, on the pressor action of exogenous Ang II. At the same time, plasma hormones and drug levels were monitored. At 1-week intervals and in a double-blind randomized fashion, 8 male volunteers received three doses of LRB081 (10, 40, and 80 mg) and placebo. Blood pressure (BP) was measured at a finger by photoplethysmograph. The peak BP response to intravenous injection of a standard dose of Ang II was determined before and for < or = 24 h after administration of an oral dose of LRB081 or placebo. After drug administration, the blood BP response to Ang II was expressed in percent of the response before drug administration. At the same time, plasma renin activity (PRA), Ang II, aldosterone, catecholamine (radioassays), and drug levels (by high-performance liquid chromatography) were monitored. After LRB081 administration, a dose dependent inhibition of the BP response to Ang II was observed. Maximal inhibition of the systolic BP response was 54 +/- 3 (mean +/- SEM), 63 +/- 2, and 93 +/- 1% with 10, 40, and 80 mg LRB081, respectively. The time to peak was 3 h for 6 subjects and 4 and 6 h for 2 others. Preliminary plasma half-life (t1/2) was calculated at 2 h. With the highest dose, the inhibition remained significant for 24 h (31 +/- 5%, p < 0.05). Maximal BP-blocking effect and maximal plasma drug level coincided, suggesting that the unmetabolized LRB081 is responsible for the antagonistic effect. PRA and Ang II increased dose dependently after LRB081 intake. Aldosterone, epinephrine, and norepinephrine concentrations remained unchanged. No clinically significant adverse reaction was observed during the study. LRB081 is a well-tolerated, orally active, potent, and long-acting Ang II receptor antagonist. Unlike in the case of losartan, no active metabolite of LRB081 has been shown to be responsible for the main effects.

Acquired antibody responses against Plasmodium vivax infection vary with host genotype for duffy antigen receptor for chemokines (DARC).

BACKGROUND: Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are 'resistant' to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens. METHODOLOGY/FINDINGS: We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion. CONCLUSION/SIGNIFICANCE: Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development.

Development of a T Cell Receptor Targeting an HLA-A*0201 Restricted Epitope from the Cancer-Testis Antigen SSX2 for Adoptive Immunotherapy of Cancer.

The clinical success of adoptive immunotherapy of cancer relies on the selection of target antigens that are highly expressed in tumor cells but absent in essential normal tissues. A group of genes that encode the cancer/testis or cancer germline antigens have been proposed as ideal targets for immunotherapy due to their high expression in multiple cancer types and their restricted expression in immunoprivileged normal tissues. In the present work we report the isolation and characterization of human T cell receptors (TCRs) with specificity for synovial sarcoma X breakpoint 2 (SSX2), a cancer/testis antigen expressed in melanoma, prostate cancer, lymphoma, multiple myeloma and pancreatic cancer, among other tumors. We isolated seven HLA-A2 restricted T cell receptors from natural T cell clones derived from tumor-infiltrated lymph nodes of two SSX2-seropositive melanoma patients, and selected four TCRs for cloning into retroviral vectors. Peripheral blood lymphocytes (PBL) transduced with three of four SSX2 TCRs showed SSX241-49 (KASEKIFYV) peptide specific reactivity, tumor cell recognition and tetramer binding. One of these, TCR-5, exhibited tetramer binding in both CD4 and CD8 cells and was selected for further studies. Antigen-specific and HLA-A*0201-restricted interferon-γ release, cell lysis and lymphocyte proliferation was observed following culture of TCR engineered human PBL with relevant tumor cell lines. Codon optimization was found to increase TCR-5 expression in transduced T cells, and this construct has been selected for development of clinical grade viral vector producing cells. The tumor-specific pattern of expression of SSX2, along with the potent and selective activity of TCR-5, makes this TCR an attractive candidate for potential TCR gene therapy to treat multiple cancer histologies.

Lipoprotein lipase and leptin are accumulated in different secretory compartments in rat adipocytes.

Adipose cells produce and secrete several physiologically important proteins, such as lipoprotein lipase (LPL), leptin, adipsin, Acrp30, etc. However, secretory pathways in adipocytes have not been characterized, and vesicular carriers responsible for the accumulation and transport of secreted proteins have not been identified. We have compared the intracellular localization of two proteins secreted from adipose cells: leptin and LPL. Adipocytes accumulate large amounts of both proteins, suggesting that neither of them is targeted to the constitutive secretory pathway. By means of velocity centrifugation in sucrose gradients, equilibrium density centrifugation in iodixanol gradients, and immunofluorescence confocal microscopy, we determined that LPL and leptin were localized in different membrane structures. LPL was found mainly in the endoplasmic reticulum with a small pool being present in low density membrane vesicles that may represent a secretory compartment in adipose cells. Virtually all intracellular leptin was localized in these low density secretory vesicles. Insulin-sensitive Glut4 vesicles did not contain either LPL or leptin. Thus, secretion from adipose cells is controlled both at the exit from the endoplasmic reticulum as well as at the level of "downstream" secretory vesicles.