Alternative academic approaches for testing homologous recombination deficiency in ovarian cancer in the MITO16A/MaNGO-OV2 trial.

BACKGROUND: The detection of homologous recombination deficiency (HRD) can identify patients who are more responsive to platinum and poly ADP ribose polymerase inhibitors (PARPi). MyChoice CDx (Myriad) is the most used HRD test in ovarian cancer (OC). However, some limitations of commercial tests exist, because of the high rate of inconclusive results, costs, and the impossibility of evaluating functional resistance mechanisms. PATIENTS AND METHODS: Two academic genomic tests and a functional assay, the RAD51 foci, were evaluated to detect HRD. One hundred patients with high-grade OC enrolled in the MITO16A/MaNGO-OV2 trial and treated with first-line therapy with carboplatin, paclitaxel, and bevacizumab were analyzed. RESULTS: The failure rate of the two genomic assays was 2%. The sensitivity in detecting HRD when compared with Myriad was 98.1% and 90.6%, respectively. The agreement rate with Myriad was 0.92 and 0.87, with a Cohen's κ coefficient corresponding to 0.84 and 0.74, respectively. For the RAD51 foci assay, the failure rate was 30%. When the test was successful, discordant results for deficient and proficient tumors were observed, and additional HRD patients were identified compared to Myriad; sensitivity was 82.9%, agreement rate was 0.65, and Cohen's κ coefficient was 0.18. The HRD detected by genomic assays and residual tumor at primary surgery and stage was correlated with progression-free survival at multivariate analysis. CONCLUSIONS: Results suggest the feasibility of academic tests for assessing HRD status that show robust concordance with Myriad and correlation with clinical outcome. The contribution of the functional information related to the RAD51 foci test to the genomic data needs further investigation.

A coiled-coil masking domain for selective activation of therapeutic antibodies.

The use of monoclonal antibodies in cancer therapy is limited by their cross-reactivity to healthy tissue. Tumor targeting has been improved by generating masked antibodies that are selectively activated in the tumor microenvironment, but each such antibody necessitates a custom design. Here, we present a generalizable approach for masking the binding domains of antibodies with a heterodimeric coiled-coil domain that sterically occludes the complementarity-determining regions. On exposure to tumor-associated proteases, such as matrix metalloproteinases 2 and 9, the coiled-coil peptides are cleaved and antigen binding is restored. We test multiple coiled-coil formats and show that the optimized masking domain is broadly applicable to antibodies of interest. Our approach prevents anti-CD3-associated cytokine release in mice and substantially improves circulation half-life by protecting the antibody from an antigen sink. When applied to antibody-drug conjugates, our masked antibodies are preferentially unmasked at the tumor site and have increased anti-tumor efficacy compared with unmasked antibodies in mouse models of cancer.

Junction opener protein increases nanoparticle accumulation in solid tumors.

Carcinomas contain tight junctions that can limit the penetration and therefore therapeutic efficacy of anticancer agents, especially those delivered by nano-carrier systems. The junction opener (JO) protein is a virus-derived protein that can transiently open intercellular junctions in epithelial tumors by cleaving the junction protein desmoglein-2 (DSG2). Co-administration of JO was previously shown to significantly increase the efficacy of various monoclonal antibodies and chemotherapy drugs in murine tumor models by allowing for increased intratumoral penetration of the drugs. To investigate the size-dependent effect of JO on nanocarriers, we used PEGylated gold nanoparticles (AuNPs) of two different sizes as model drugs and investigated their biodistribution following JO protein treatment. By inductively coupled plasma mass spectrometry (ICP-MS), JO was found to significantly increase bulk tumor accumulation of AuNPs of 35nm but not 120nm particles in both medium (200-300mm3) and large (500-600mm3) tumors. Image analysis of tumor sections corroborates this JO-mediated increase in tumor accumulation of AuNPs. Quantitative intratumoral distribution analyses show that most nanoparticles were found within 100µm of the vasculature, and that the penetration profiles of AuNPs are not significantly affected by JO treatment at the 6h timepoint.

Virus-Inspired Polymer for Efficient In†Vitro and In†Vivo Gene Delivery.

Clinical translation of nucleic acids drugs has been stunted by limited delivery options. Herein, we report a synthetic polymer designed to mimic viral mechanisms of delivery called VIPER (virus-inspired polymer for endosomal release). VIPER is composed of a polycation block for condensation of nucleic acids, and a pH-sensitive block for acid-triggered display of a lytic peptide to promote trafficking to the cell cytosol. VIPER shows superior efficiencies compared to commercial agents when delivering genes to multiple immortalized cell lines. Importantly, in murine models, VIPER facilitates effective gene transfer to solid tumors.

Safety analysis, association with response and previous treatments of everolimus and exemestane in 181 metastatic breast cancer patients: A multicenter Italian experience.

PURPOSE: The everolimus and exemestane combination represents a treatment option for the endocrine sensitive metastatic breast cancer (MBC) patients. The toxicity profile reported in the Bolero 2 trial showed the feasibility in the selected patients. Few data are available for the unselected population. METHODS: In order to evaluate the safety in the unselected population of the clinical practice and to evaluate a possible association of toxicities with previous treatments, clinical data from 181 consecutive patients were retrospectively collected. RESULTS: Due to toxic events, everolimus dosage was reduced to 5 mg in 27% of patients. No association was found in the analysis between toxicity and number of prior therapies, neither between toxicity and response. In the multivariate analysis the previous exposure to anthracyclines for advanced disease represents the only predictive factor of developing grade ≥2 toxicity (OR = 2.85 CI 95% 1.07-7.59, p = 0.036). CONCLUSIONS: The association of everolimus and exemestane has confirmed to be a safe and effective treatment for endocrine sensitive MBC patients even in routine clinical practice. The rate of treatment discontinuation due to toxicity is low and none association between previous number of treatments and response or between toxicity and response was found.

Oocyte quality of tambaqui (Colossoma macropomum) during the reproductive season.

The study aimed to analyze the Colossoma macropomum reproductive behavior and quality of the female gametes throughout the reproductive season. The experiment was carried out in Pimenta Bueno - Rondônia State (Northern Brazil) during the reproductive season (2010-2011) using 36 females. Each sampling was performed on a 15 ± 5 days interval. Female gametes were collected by stripping and the following analyses were performed: weight of oocytes released (g); productivity index, fertilization and hatching rate. During the sampling period was verified effect (p < 0.05) of collecting time into the season for oocytes weight, productivity index and fertilization rate. Although the period 3 (December) did not differ significantly from other periods, it showed better parameters for the quality of C. macropomum oocytes.

Heterogeneity of KRAS, NRAS, BRAF and PIK3CA mutations in metastatic colorectal cancer and potential effects on therapy in the CAPRI GOIM trial.

BACKGROUND: Evidence suggests that metastatic colorectal carcinoma (mCRC) has a high level of intratumor heterogeneity. We carried out a quantitative assessment of tumor heterogeneity for KRAS, NRAS, BRAF and PIK3CA mutations, in order to assess potential clinical implications. PATIENTS AND METHODS: Tumor samples (n = 182) from the CAPRI-GOIM trial of first-line cetuximab + FOLFIRI in KRAS exon-2 wild-type mCRC patients were assessed by next-generation sequencing that allows quantitative assessment of mutant genes. Mutant allelic frequency was normalized for the neoplastic cell content and, assuming that somatic mutations usually affect one allele, the Heterogeneity Score (HS) was calculated by multiplying by 2 the frequency of mutant alleles in neoplastic cells. Therefore, HS virtually corresponds to the fraction of neoplastic cells carrying a specific mutation. RESULTS: The KRAS HS ranged between 12 and 260 with mean value of 87.1 and median value of 84.4, suggesting that in most CRC, the majority of neoplastic cells carry mutant KRAS. Similar findings were observed for NRAS (HS range 35.5-146.7; mean 102.8; median 117.1). In contrast, in BRAF (HS range 17.1-120; mean 54.8; median 54.3) and PIK3CA (HS range 14.3-120; mean 59.5; median 47.3) mutant cases, only a fraction of neoplastic cells seem to carry the mutant allele. The response rate was 70% in KRAS mutant patients with an HS <33 (low KRAS; n = 10) and 45.7% in KRAS HS >33 patients (high KRAS; n = 35); median progression-free survival were 7.97 and 8.37 months, respectively. Low-KRAS tumors had a higher frequency of additional mutations in PIK3CA when compared with high-KRAS (6/10 versus 8/35). CONCLUSIONS: KRAS and NRAS mutations are usually present in the majority of neoplastic cells, whereas BRAF and PIK3CA mutations often affect a limited fraction of transformed cells. Resistance to cetuximab in low-KRAS patients might be driven by the complex mutational profile rather than KRAS mutation load.

Expression and functional role of CRIPTO-1 in cutaneous melanoma.

BACKGROUND: CRIPTO-1 (CR-1) is involved in the pathogenesis and progression of human carcinoma of different histological origin. In this study we addressed the expression and the functional role of CR-1 in cutaneous melanoma. METHODS: Expression of CR-1 protein in melanomas and melanoma cell lines was assessed by immunohistochemistry, western blotting and/or flow cytometry. Levels of mRNA were evaluated by real-time PCR. Invasion assays were performed in Matrigel-coated modified Boyden chambers. RESULTS: Expression of CR-1 protein and/or mRNA was found in 16 out of 37 primary human cutaneous melanomas and in 12 out of 21 melanoma cell lines. Recombinant CR-1 protein activated in melanoma cells c-Src and, at lesser extent, Smad signalling. In addition, CR-1 significantly increased the invasive ability of melanoma cells that was prevented by treatment with either the ALK4 inhibitor SB-431542 or the c-Src inhibitor saracatinib (AZD0530). Anti-CR-1 siRNAs produced a significant inhibition of the growth and the invasive ability of melanoma cells. Finally, a close correlation was found in melanoma cells between the levels of expression of CR-1 and the effects of saracatinib on cell growth. CONCLUSION: These data indicate that a significant fraction of cutaneous melanoma expresses CR-1 and that this growth factor is involved in the invasion and proliferation of melanoma cells.

Produção animal e valor nutritivo da forragem de pastagem de coastcross consorciada com amendoim forrageiro / Animal production and nutritive value of a coastcross pasture mixed with forage peanut

Neste estudo foram utilizados os tratamentos: coastcross + amendoim forrageiro + 200kg/ha de N; coastcross + amendoim forrageiro + 100kg/ha de N; coastcross + 200kg/ha de N e coastcross + amendoim forrageiro (parcelas) no inverno, primavera, verão e outono (sub parcelas), delineados em blocos ao acaso. Novilhas foram manejadas sob lotação contínua e taxa de lotação variável em pastagem mantida a 17cm de altura. Amostras foram coletadas a cada 28 dias para determinar o valor nutritivo da forragem. Foram avaliados: ganho médio diário (GMD), ganho de peso vivo (GPV), taxa de lotação (TL) e número de animais dia (NAD). Quanto ao valor nutritivo da forragem, os piores resultados ocorreram nas pastagens sem adubação, 16,9 por cento e 6,0 por cento de PB de folha e colmo, respectivamente, e 70,1 por cento de FDN de folha. Na primavera e no verão, o GMD foi mais alto, 0,518 e 0,515kg/animal do que no inverno e outono, 0,396 e 0,293kg/animal, respectivamente. A TL foi superior nas pastagens que receberam a maior dose de nitrogênio, 5,38UA/ha em média, e no verão, 6,81UA/ha. O GPV foi mais elevado nas áreas com adubação, 1341kg de PV/ha, em relação aos pastos não adubados, 735kg/ha.

In this study, the ollowing treatments were used: coastcross + forage peanut + 200kg/ha of N; coastcross + forage peanut + 100kg/ha of N; coastcross + 200kg/ha of N and coastcross + forage peanut (plots) in the winter, spring, summer, and autumn (subplots), designed in randomized blocks. Heifers were managed under continuous stocking and variable stocking rate on pasture maintained at 17cm height. Samples were collected every 28 days determining the nutritional value of forage. Average daily gain (ADG), weight gain (WG), stocking rate (SR), and number of animals/day (NAD) were evaluated. As for forage nutritional value, the worst results were found in pasture without fertilization, 16.9 percent and 6.0 percent CP of leaf and stem, respectively, and 70.1 percent NDF in leaves. In the spring and summer, animals had a higher ADG, 0.518 and 0.515kg/animal, than 0.396 and 0.293kg/animal in the winter and autumn, respectively. SR was higher in pastures that received higher nitrogen doses, 5.38AU/ha in average; and in the summer, 6.81AU/ha. LWG was higher in fertilized areas, 1,341kg LW/ha, than in not fertilized pastures, 735kg/ha.

Produção e qualidade do pasto de coastcross consorciado ou não com amendoim forrageiro com ou sem aplicação de nitrogênio / Production and quality of coastcross pasture associated or not with forage peanut with or without nitrogen

Avaliaram-se a taxa de acúmulo foliar (TAF), a produção total de forragem (PTF), a composição química da forragem e os valores de digestibilidade in vitro da matéria seca (DIVMS) do coastcross (Cynodon dactylon) e do amendoim forrageiro (Arachis pintoi) em pastagem consorciada. Foram avaliados os tratamentos (parcelas): coastcross + A. pintoi sem nitrogênio (CA0); coastcross + A. pintoi com 100kg de nitrogênio (CA100); coastcross com 200kg de nitrogênio (C200) e coastcross + A. pintoi com 200kg de nitrogênio (CA200), e as estações (subparcelas) do ano inverno, primavera, verão e outono. Utilizou-se um delineamento experimental em blocos ao acaso, em esquema de parcelas subdivididas no tempo, com duas repetições. Os tratamentos com os maiores níveis de adubação de nitrogênio apresentaram TAF mais alto que os demais (P<0,05). No verão ocorreu a maior PTF, seguida da primavera, outono e inverno, mas estes não diferiram entre si. Não houve diferença (P>0,05) entre os tratamentos para as variáveis proteína bruta, fibra em detergente neutro e digestibilidade in vitro de matéria seca, tanto para as frações de folhas do coastcross, como para a planta inteira do A. pintoi. A adubação nitrogenada, quando aplicada à pastagem de coastcross singular ou associada ao amendoim forrageiro, proporcionou aumento da TAF e da PTF, com maior produtividade e qualidade no verão.

Forage accumulation rate (FAR), total forage production (TFP), chemical composition and in vitro dry matter digestibility (IVDMD) of coastcross pasture mixed with Arachis pintoi (AP) were evaluated. Treatments plots were: coastcross + A. pintoi without N (CA0); coastcross + A. pintoi with 100kg of N (CA100); coastcross with 200kg of N (C200); and coastcross + A. pintoi with 200kg of N (CA200); and seasons of the year (split-plots): winter, spring, summer, and autumn were analyzed. A randomized block design was used subdivided into time plots, with two replications. Treatments with the highest fertilizer levels presented higher FAR than others (P<0.05), with higher TFP during the summer, followed by spring, autumn, and winter, with no differences among then. There were no differences (P>0.05) among treatments for CP, NDF, and IVDMD on coastcross leaf fractions and Arachis pintoi whole plant. Nitrogen fertilization, when applied to single coastcross pasture or mixed with forage peanut, increased accumulation rate and forage accumulation, with higher productivity and quality in summer.

Role of cellular heparan sulfate proteoglycans in infection of human adenovirus serotype 3 and 35.

Species B human adenoviruses (Ads) are increasingly associated with outbreaks of acute respiratory disease in U.S. military personnel and civil population. The initial interaction of Ads with cellular attachment receptors on host cells is via Ad fiber knob protein. Our previous studies showed that one species B Ad receptor is the complement receptor CD46 that is used by serotypes 11, 16, 21, 35, and 50 but not by serotypes 3, 7, and 14. In this study, we attempted to identify yet-unknown species B cellular receptors. For this purpose we used recombinant Ad3 and Ad35 fiber knobs in high-throughput receptor screening methods including mass spectrometry analysis and glycan arrays. Surprisingly, we found that the main interacting surface molecules of Ad3 fiber knob are cellular heparan sulfate proteoglycans (HSPGs). We subsequently found that HSPGs acted as low-affinity co-receptors for Ad3 but did not represent the main receptor of this serotype. Our study also revealed a new CD46-independent infection pathway of Ad35. This Ad35 infection mechanism is mediated by cellular HSPGs. The interaction of Ad35 with HSPGs is not via fiber knob, whereas Ad3 interacts with HSPGs via fiber knob. Both Ad3 and Ad35 interacted specifically with the sulfated regions within HSPGs that have also been implicated in binding physiologic ligands. In conclusion, our findings show that Ad3 and Ad35 directly utilize HSPGs as co-receptors for infection. Our data suggest that adenoviruses evolved to simulate the presence of physiologic HSPG ligands in order to increase infection.

[Complete atrioventricular block in Kearns-Sayre syndrome]. / Bloqueio atrioventricular total em síndrome de Kearns-Sayre.

A thirty-three year old woman, known to have Kearns-Sayre syndrome for eight years, had an ECG pattern of right bundle branch block and left anterior fascicular block that evolved to complete atrioventricular block, leading her to a syncopal episode. A temporary pacemaker and a permanent one were installed. The patient has been asymptomatic so far.