Analysis of Circulating Tumor Cells in Ovarian Cancer and Their Clinical Value as a Biomarker

Background/Aims: Monitoring the appearance and progression of tumors are important for improving the survival rate of patients with ovarian cancer. This study aims to examine circulating tumor cells (CTCs) in epithelial ovarian cancer (EOC) patients to evaluate their clinical significance in comparison to the existing biomarker CA125. Methods: Immuomagnetic bead screening, targeting epithelial antigens on ovarian cancer cells, combined with multiplex reverse transcriptase-polymerase chain reaction (Multiplex RT-PCR) was used to detect CTCs in 211 samples of peripheral blood (5 ml) from 109 EOC patients. CTCs and CA125 were measured in serial from 153 blood and 153 serum samples from 51 patients and correlations with treatment were analyzed. Immunohistochemistry was used to detect the expression of tumor-associated proteins in tumor tissues and compared with gene expression in CTCs from patients. Results: CTCs were detected in 90% (98/109) of newly diagnosed patients. In newly diagnosed patients, the number of CTCs was correlated with stage (p=0.034). Patients with stage IA-IB disease had a CTC positive rate of 93% (13/14), much higher than the CA125 positive rate of only 64% (9/14) for the same patients. The numbers of CTCs changed with treatment, and the expression of EpCAM (p=0.003) and HER2 (p=0.035) in CTCs was correlated with resistance to chemotherapy. Expression of EpCAM in CTCs before treatment was also correlated with overall survival (OS) (p=0.041). Conclusion: Detection of CTCs allows early diagnose and expression of EpCAM in CTC positive patients predicts prognosis and should be helpful for monitoring treatment

Numbers of <i>Retn</i> and <i>Retnl</i> genes found in groups of vertebrate species.

<p>¹ Number of species examined / number of species with a <i>Retn</i> or <i>Retnl</i> gene / number of species with at least one intact <i>Retn</i> or <i>Retnl</i> coding sequence for each group.</p><p>² Genomic sequences that fail to predict part of a <i>Retn</i> or <i>Retnl</i> gene sequence, possibly due to a gap in the genome assembly.</p><p>³ Sequence contains mutations that cause a frame shift or introduce premature stop codons in the <i>Retn</i> or <i>Retnl</i> coding sequence.</p><p>Numbers of <i>Retn</i> and <i>Retnl</i> genes found in groups of vertebrate species.</p

Evolution of the Vertebrate Resistin Gene Family

<div><p>Resistin (encoded by <i>Retn</i>) was previously identified in rodents as a hormone associated with diabetes; however human resistin is instead linked to inflammation. Resistin is a member of a small gene family that includes the resistin-like peptides (encoded by <i>Retnl</i> genes) in mammals. Genomic searches of available genome sequences of diverse vertebrates and phylogenetic analyses were conducted to determine the size and origin of the resistin-like gene family. Genes encoding peptides similar to resistin were found in Mammalia, Sauria, Amphibia, and Actinistia (coelacanth, a lobe-finned fish), but not in Aves or fish from Actinopterygii, Chondrichthyes, or Agnatha. <i>Retnl</i> originated by duplication and transposition from <i>Retn</i> on the early mammalian lineage after divergence of the platypus, but before the placental and marsupial mammal divergence. The resistin-like gene family illustrates an instance where the locus of origin of duplicated genes can be identified, with <i>Retn</i> continuing to reside at this location. Mammalian species typically have a single copy <i>Retn</i> gene, but are much more variable in their numbers of <i>Retnl</i> genes, ranging from 0 to 9. Since <i>Retn</i> is located at the locus of origin, thus likely retained the ancestral expression pattern, largely maintained its copy number, and did not display accelerated evolution, we suggest that it is more likely to have maintained an ancestral function, while <i>Retnl</i>, which transposed to a new location, displays accelerated evolution, and shows greater variability in gene number, including gene loss, likely evolved new, but potentially lineage-specific, functions.</p></div

Alignment of Retn and Retnl protein sequences.

<p>Alignment of predicted resistin and resistin-like protein sequences from human, mouse, opossum, Anole lizard, and coelacanth (lobe-finned fish). The human Retn sequence is shown at the top in single letter code. Dots in the alignments represent identity to the human Retn sequence, with differences indicated in single letter code. Asterisks below the alignment identify residues that are perfectly conserved among all of the selected sequences.</p

Genomic organization of genes near <i>Retn</i> genes of representative vertebrate species.

<p><i>Retn</i> genes are labeled in red. Genes that share genomic location with human genes are labeled in yellow, while genes labeled in green are either lineage-specific genes or are found at a different genomic location in the human genome (genes without name do not have a human ortholog). The painted turtle genomic neighborhood is composed of two scaffolds that are likely adjacent. Chromosome, genomic scaffold, or sequence accession numbers, with approximate coordinates and size, of the displayed fragment are shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130188#pone.0130188.s009" target="_blank">S1 Table</a> for details on genomic locations of <i>Retn</i> genes. Gene sizes and distances between genes are not to scale. Arrowheads indicate direction of transcription. Gene symbols are: <i>Retn</i>, resistin; <i>Xab2</i>, XPA binding protein 2; <i>Pet100</i>, PET100 homolog; <i>Pcp2</i>, Purkinje cell protein 2; <i>Stxbp2</i>, Syntaxin binding protein 2; <i>Mcemp1</i>, Mast cell-expressed membrane protein 1; <i>Trappc5</i>, Trafficking protein particle complex 5; <i>Fcer2</i>, Fc fragment of IgE, low affinity II, receptor for (CD23); <i>Clec4g</i>, C-type lectin domain family 4, member G; <i>Kiss1r</i>, KISS1 receptor; <i>Jrk</i>, Jerky; <i>Pglyrp6</i>, Peptidoglycan recognition protein 6; <i>Rasal3</i>, RAS protein activator like 3; <i>Emr</i>-like, Egf-like module containing, mucin-like, hormone receptor-like; <i>Wiz</i>, Widely interspaced zinc finger motifs; and <i>Fam102b</i>, Family with sequence similarity 102, member B.</p

Model for the evolution of the resistin and resistin-like genes.

<p><i>Retn</i> and <i>Retnl</i> genes are indicated by the red arrows, with the arrow pointing in the direction of transcription. Other genes are shown in yellow (locus of origin) or blue (location of inserted <i>Retnl</i> gene). Curved arrows indicated gene duplications that were either a transposition (to generate <i>Retnl</i>) or tandem, on the rodent and primate lineages. X’s indicate inactivating mutations in the primate and artiodactyl genes that generate pseudogenes. (A). In the ancestor to mammals, <i>Retn</i> was located in the locus of origin. (B). On an early mammalian lineage, prior to the placental mammal-marsupial divergence, a copy of <i>Retn</i> was transposed to a new genomic location to generate the <i>Retnl</i> gene. The transposition likely allowed <i>Retnl</i> to acquire a novel expression pattern. (C). <i>Retn</i> and <i>Retnl</i> genes have different fates on divergent mammalian lineages. While <i>Retn</i> remained as a single copy intact gene on different mammalian lineages, <i>Retnl</i> had different fates, raising the possibility that it acquired lineage-specific functions. <i>Retnl</i> remained as a single copy gene in perissodactyls, duplicated intact (potentially functional) copies in rodents, duplicated and generated a pseudogene in primates, or was inactivated in artiodactyls.</p

Genomic organization of genes near <i>Retnl</i> genes of representative vertebrate species.

<p><i>Retnl</i> genes are labeled in red. Genes that share genomic location with human genes are labeled in black, while genes labeled in green are either lineage-specific genes or are found at a different genomic location in the human genome (genes without names do not have a human ortholog). Chromosome, genomic scaffold, or sequence accession numbers, with approximate coordinates and size, of the displayed fragment is shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130188#pone.0130188.s009" target="_blank">S1 Table</a> for details on genomic locations of <i>Retnl</i> genes. Gene sizes and distances between genes are not to scale. Arrowheads indicate direction of transcription. Gene symbols are: <i>Retnl</i>, resistin-like; <i>Morc1</i>, MORC family CW-type zinc finger 1; <i>Guca1c</i>, Guanylate cyclase activator 1C; <i>Trat1</i>, T cell receptor associated transmembrane adaptor 1; <i>Dzip3</i>, DAZ interacting zinc finger protein 3; <i>Kiaa1524</i>, KIAA1524; <i>Myh15</i>, Myosin, heavy chain 15; <i>Hhla2</i>, HERV-H LTR-associating 2; <i>Hjurp</i>, Holliday junction recognition protein; <i>Sh2d1b</i>, SH2 domain containing 1B; <i>Ift57</i>, Intraflagellar transport 57; and <i>Mrps23</i>, Mitochondrial ribosomal protein S23.</p

Phylogeny of <i>Retn</i> and <i>Retnl</i> genes.

<p>Phylogeny inferred by the Bayesian method for 136 <i>Retn</i> and <i>Retnl</i> sequences from diverse vertebrates. The phylogeny was rooted with the Coelacanth <i>Retn</i> sequence. Similar phylogenies were obtained if sequences from Sauria (coelacanth sequence not used) were used as the outgroup or if other methods were used (e.g., see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130188#pone.0130188.s006" target="_blank">S6 Fig</a>). Numbers at the nodes indicate posterior probabilities, with those for the nodes in early mammalian evolution shown in bold. Branch lengths are proportional to the inferred amount of change, with the scale bar at the bottom. Diamonds indicate inferred gene duplication events. <i>Retnl</i> genes are shown in the upper part of the tree while <i>Retn</i> genes are below.</p

Evolution of Hepatic Glucose Metabolism: Liver-Specific Glucokinase Deficiency Explained by Parallel Loss of the Gene for Glucokinase Regulatory Protein (GCKR)

<div><p>Background</p><p>Glucokinase (GCK) plays an important role in the regulation of carbohydrate metabolism. In the liver, phosphorylation of glucose to glucose-6-phosphate by GCK is the first step for both glycolysis and glycogen synthesis. However, some vertebrate species are deficient in GCK activity in the liver, despite containing <i>GCK</i> genes that appear to be compatible with function in their genomes. Glucokinase regulatory protein (GCKR) is the most important post-transcriptional regulator of GCK in the liver; it participates in the modulation of GCK activity and location depending upon changes in glucose levels. In experimental models, loss of GCKR has been shown to associate with reduced hepatic GCK protein levels and activity.</p> <p>Methodology/Principal Findings</p><p><i>GCKR</i> genes and <i>GCKR</i>-like sequences were identified in the genomes of all vertebrate species with available genome sequences. The coding sequences of <i>GCKR</i> and <i>GCKR</i>-like genes were identified and aligned; base changes likely to disrupt coding potential or splicing were also identified.</p> <p>Conclusions/Significance</p><p><i>GCKR</i> genes could not be found in the genomes of 9 vertebrate species, including all birds. In addition, in multiple mammalian genomes, whereas <i>GCKR</i>-like gene sequences could be identified, these genes could not predict a functional protein. Vertebrate species that were previously reported to be deficient in hepatic GCK activity were found to have deleted (birds and lizard) or mutated (mammals) <i>GCKR</i> genes. Our results suggest that mutation of the <i>GCKR</i> gene leads to hepatic GCK deficiency due to the loss of the stabilizing effect of GCKR.</p> </div

Relative rate tests.

1<p>– Number of unique amino acid substitutions on the lineages to species A and B when the Tasmanian devil sequence was used as the outgroup.</p>2<p>– Chi square value for the expectation that an equal number of substitutions occurred on each lineage.</p