HER2 overexpression and amplification is present in a subset of ovarian mucinous carcinomas and can be targeted with trastuzumab therapy

<p>Abstract</p> <p>Background</p> <p>The response rate of ovarian mucinous carcinomas to paclitaxel/carboplatin is low, prompting interest in targeted molecular therapies. We investigated HER2 expression and amplification, and the potential for trastuzumab therapy in this histologic subtype of ovarian cancer.</p> <p>Methods</p> <p>HER2 status was tested in 33 mucinous carcinomas and 16 mucinous borderline ovarian tumors (BOT)). Five cases with documented recurrence and with tissue from the recurrence available for testing were analyzed to determine whether HER2 amplification status changed over time. Three prospectively identified recurrent mucinous ovarian carcinomas were assessed for HER2 amplification and patients received trastuzumab therapy with conventional chemotherapy.</p> <p>Results</p> <p>Amplification of HER2 was observed in 6/33 (18.2%) mucinous carcinomas and 3/16 (18.8%) BOT. HER2 amplification in primary mucinous carcinomas was not associated with an increased likelihood of recurrence. The prospectively identified recurrent mucinous carcinomas showed overexpression and amplification of HER2; one patient's tumor responded dramatically to trastuzumab in combination with conventional chemotherapy, while another patient experienced an isolated central nervous system recurrence after trastuzumab therapy.</p> <p>Conclusion</p> <p>HER2 amplification is relatively common in ovarian mucinous carcinomas (6/33, 18.2%), although not of prognostic significance. Trastuzumab therapy is a treatment option for patients with mucinous carcinoma when the tumor has HER2 amplification and overexpression.</p

Establishment and characterization of UM-EC-2, a tamoxifen-sensitive, estrogen receptor-negative human endometrial carcinoma cell line,

UM-EC-2 was established from a patient with poorly differentiated stage IB endometrial carcinoma. This cell line produces tumors in nude mice that have the same histological features as the patient's tumor. UM-EC-2 cells express [beta]2-microglobulin, the epidermal growth factor receptor (EGF), and the H blood group antigen. This membrane antigen phenotype is consistent with cells of human endometrial origin. The karyotype of UM-EC-2 is fairly complex, with rearrangements affecting all chromosomes except 3, 10, 14, 19, and 20. There were two populations of cells, a hyperdiploid population with a modal number of 53-55 and a hypertetraploid population with a modal number of 109. A postulated sequence of events before and after tetraploidization is suggested based on the number of copies of individual chromosomes and rearrangements. Comparison of the UM-EC-2 karyotype to that of UM-EC-1 (a previously described line from a different patient with endometrial carcinoma) revealed that the two lines share eight very similar chromosome changes, which include loss of most of chromosome 4, breakpoints affecting proximal bands on 8p, loss of most of 9q, a breakpoint at 12q22, loss of 13q, breakpoints in proximal bands on 18q, and a breakpoint at 22p11. These changes may represent nonrandom chromosome abnormalities in poorly differentiated endometrial cancer. Estrogen (ER) and progesterone (PgR) receptors were not detected in either the primary tumor or the cell line. Nevertheless, UM-EC-2 cells were very sensitive to growth inhibition by tamoxifen (TAM) in vitro. One micromolar TAM caused 50% inhibition of cell growth, 2.5 [mu]M caused cytostasis, and 5 [mu]M TAM was cytotoxic, killing all cells after 5-7 days of exposure to the drug. Paradoxically, 100 nM estradiol (E2) caused a moderate increase in the growth of the cells but it did not prevent or reverse growth inhibitory effects of TAM. These findings support the concept that in some tumors TAM causes growth inhibition by an ER-independent mechanism. UM-EC-2 cells were also sensitive to growth regulation by EGF. Thus, these cells provide a new in vitro model of human endometrial cancer in which the roles of both TAM and EGF as growth regulatory substances can be investigated.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28594/1/0000402.pd

The Trypanosoma cruzi Virulence Factor Oligopeptidase B (OPBTc) Assembles into an Active and Stable Dimer

Oligopeptidase B, a processing enzyme of the prolyl oligopeptidase family, is considered as an important virulence factor in trypanosomiasis. Trypanosoma cruzi oligopeptidase B (OPBTc) is involved in host cell invasion by generating a Ca2+-agonist necessary for recruitment and fusion of host lysosomes at the site of parasite attachment. The underlying mechanism remains unknown and further structural and functional characterization of OPBTc may help clarify its physiological function and lead to the development of new therapeutic molecules to treat Chagas disease. In the present work, size exclusion chromatography and analytical ultracentrifugation experiments demonstrate that OPBTc is a dimer in solution, an association salt and pH-resistant and independent of intermolecular disulfide bonds. The enzyme retains its dimeric structure and is fully active up to 42°C. OPBTc is inactivated and its tertiary, but not secondary, structure is disrupted at higher temperatures, as monitored by circular dichroism and fluorescence spectroscopy. It has a highly stable secondary structure over a broad range of pH, undergoes subtle tertiary structure changes at low pH and is less stable under moderate ionic strength conditions. These results bring new insights into the structural properties of OPBTc, contributing to future studies on the rational design of OPBTc inhibitors as a promising strategy for Chagas disease chemotherapy

Global genome diversity of the Leishmania donovani complex.

Protozoan parasites of the Leishmania donovani complex - L. donovani and L. infantum - cause the fatal disease visceral leishmaniasis. We present the first comprehensive genome-wide global study, with 151 cultured field isolates representing most of the geographical distribution. L. donovani isolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority of L. infantum samples fell into one globally-distributed group with little diversity. This picture is complicated by several hybrid lineages. Identified genetic groups vary in heterozygosity and levels of linkage, suggesting different recombination histories. We characterise chromosome-specific patterns of aneuploidy and identified extensive structural variation, including known and suspected drug resistance loci. This study reveals greater genetic diversity than suggested by geographically-focused studies, provides a resource of genomic variation for future work and sets the scene for a new understanding of the evolution and genetics of the Leishmania donovani complex

Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage

Biochemical and Functional Characterization of Serine Proteases in Leishmania

Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites and in the host-parasite relationship. They are important targets for the development of new anti-parasitic therapy. Protozoan parasites like Leishmania predominantly express Clan CA cysteine proteases. It was therefore unexpected to find a high level of serine protease activity in Leishmania donovani. Oligopeptidase B (OPB) was identified as the responsible enzyme. This was confirmed by OPB gene knockout, which resulted in the disappearance of the major serine protease activity. To delineate the role of OPB in parasite physiology, two-dimensional gel electrophoresis was carried out on OPB -/- versus wildtype parasites. Four protein species were significantly elevated in OPB -/- parasites, and all four were identified as enolase. Aside from its classic role in carbohydrate metabolism, enolase is known to bind plasminogen and function as a virulence factor for several infectious microbes. As expected, there was a striking alteration in macrophage responses to Leishmania when OPB was deleted. While wildtype parasites elicited little response from infected macrophages, OPB -/- parasites induced a massive upregulation in transcription. OPB -/- parasites displayed decreased virulence in the murine infection model. Additionally we have investigated the subtilisin (SUB) of Leishmania because it is a single-copy gene and subtilisins play important roles in virulence of other protozoa. Leishmania subtilisin was deleted by gene knockout, which resulted in reduced ability to undergo promastigote-amastigote differentiation in vitro. EM of SUB knockout amastigotes revealed abnormal membrane structures, retained flagella, and binucleation. SUB deficient Leishmania displayed reduced virulence in hamster and murine infection models. Histology of spleens from SUB knockout-infected hamsters revealed the absence of psammoma body calcifications. To delineate the role of SUB in parasite physiology, two-dimensional gel electrophoresis was carried out on SUB -/- versus wildtype parasites. SUB knockout parasites showed altered regulation of the trypanothione reductase system. Trypanosomatids lack glutathione reductase, and rely on the novel trypanothione reductase system to detoxify ROSs and maintain redox homeostasis. The predominant tryparedoxin peroxidase was decreased in SUB -/- parasites, and higher molecular weight isoforms of the enzyme were present, suggesting altered processing

Experience with low-dose replacement therapy in the initial management of severe pediatric acquired primary hypothyroidism.

Rapid hormonal replacement of children with severe primary hypothyroidism frequently results in irritability and poor concentration. To alleviate these problems we have been using initial low-dose thyroxine treatment, building up to a final dose in an incremental manner over 4 1/2 to 6 months. Because of concern this regimen may compromise growth, we reviewed our experience treating 14 children and adolescents. For the 10 patients with remaining growth potential, 5 to 7 month growth velocity from the onset of treatment was 8.5 +/- 1.9 cm/year (range 5.7-10.9), and 5 to 7 month growth velocity z-score 1.5 +/- 1.7 (range 0.2-4.9). For the entire group, the thyroxine dose required to normalize TSH was 1.6 +/- 0.74 microgram/kg (range 0.9-3.4) or 60.7 +/- 18.9 micrograms/m2 (range 37.5-97.7). Based on the 5 to 7 month z-score, we conclude that satisfactory growth can be achieved on this regimen despite biochemical hypothyroidism. Thyroxine doses required to induce initial euthyroidism are lower than previously proposed