Early diagnosis of pancreatic cancer: neutrophil gelatinase-associated lipocalin as a marker of pancreatic intraepithelial neoplasia

Pancreatic cancer is a highly lethal malignancy with a dismal 5-year survival of less than 5%. The scarcity of early biomarkers has considerably hindered our ability to launch preventive measures for this malignancy in a timely manner. Neutrophil gelatinase-associated lipocalin (NGAL), a 24-kDa glycoprotein, was reported to be upregulated nearly 27-fold in pancreatic cancer cells compared to normal ductal cells in a microarray analysis. Given the need for biomarkers in the early diagnosis of pancreatic cancer, we investigated the expression of NGAL in tissues with the objective of examining if NGAL immunostaining could be used to identify foci of pancreatic intraepithelial neoplasia, premalignant lesions preceding invasive cancer. To examine a possible correlation between NGAL expression and the degree of differentiation, we also analysed NGAL levels in pancreatic cancer cell lines with varying grades of differentiation. Although NGAL expression was strongly upregulated in pancreatic cancer, and moderately in pancreatitis, only a weak expression could be detected in the healthy pancreas. The average composite score for adenocarcinoma (4.26±2.44) was significantly higher than that for the normal pancreas (1.0) or pancreatitis (1.0) (P<0.0001). Further, although both well- and moderately differentiated pancreatic cancer were positive for NGAL, poorly differentiated adenocarcinoma was uniformly negative. Importantly, NGAL expression was detected as early as the PanIN-1 stage, suggesting that it could be a marker of the earliest premalignant changes in the pancreas. Further, we examined NGAL levels in serum samples. Serum NGAL levels were above the cutoff for healthy individuals in 94% of pancreatic cancer and 62.5% each of acute and chronic pancreatitis samples. However, the difference between NGAL levels in pancreatitis and pancreatic cancer was not significant. A ROC curve analysis revealed that ELISA for NGAL is fairly accurate in distinguishing pancreatic cancer from non-cancer cases (area under curve=0.75). In conclusion, NGAL is highly expressed in early dysplastic lesions in the pancreas, suggesting a possible role as an early diagnostic marker for pancreatic cancer. Further, serum NGAL measurement could be investigated as a possible biomarker in pancreatitis and pancreatic adenocarcinoma

Astrocytes grown in Alvetex® 3 dimensional scaffolds retain a non-reactive phenotype

yesProtocols which permit the extraction of primary astrocytes from either embryonic or postnatal mice are well established however astrocytes in culture are different to those in the mature CNS. Three dimensional (3D) cultures, using a variety of scaffolds may enable better phenotypic properties to be developed in culture. We present data from embryonic (E15) and postnatal (P4) murine primary cortical astrocytes grown on coated coverslips or a 3D polystyrene scaffold, Alvetex. Growth of both embryonic and postnatal primary astrocytes in the 3D scaffold changed astrocyte morphology to a mature, protoplasmic phenotype. Embryonic-derived astrocytes in 3D expressed markers of mature astrocytes, namely the glutamate transporter GLT-1 with low levels of the chondroitin sulphate proteoglycans, NG2 and SMC3. Embroynic astrocytes derived in 3D show lower levels of markers of reactive astrocytes, namely GFAP and mRNA levels of LCN2, PTX3, Serpina3n and Cx43. Postnatal-derived astrocytes show few protein changes between 2D and 3D conditions. Our data shows that Alvetex is a suitable scaffold for growth of astrocytes, and with appropriate choice of cells allows the maintenance of astrocytes with the properties of mature cells and a non-reactive phenotype.BBSR

Expression of a mouse replacement histone H3.3 gene with a highly conserved 3' noncoding region during SV40- and polyoma-induced Go to S-phase transition.

We have isolated and sequenced a mouse replacement variant histone H3.3 cDNA. It corresponds to the most abundant mRNA expressed from a unique gene by the use of one out of three polyadenylation sites. The 3' non coding region of H3.3 is very long (approximately 1100 nt) and highly conserved throughout evolution since it is about 95% homologous to the 3' non coding region of the chicken H3.3B gene. We studied the expression of the H3.3 gene during SV40- and polyoma-induced mitotic host reaction in confluent, Go-arrested primary mouse kidney cell cultures. H3.3 replacement variant mRNA steady state levels increased during the Go to S-phase transition, apparently as the result of two mechanisms: one related to cell growth, whereas the other was linked to cellular DNA synthesis. The latter mechanism was however far less pronounced than with replication histone variant mRNAs. The biological implications of these results are discussed

Function of the Evx-2 gene in the morphogenesis of vertebrate limbs

Vertebrate gene members of the HoxD complex are essential for proper development of the appendicular skeletons. Inactivation of these genes induces severe alterations in the size and number of bony elements. Evx-2, a gene related to the Drosophila even-skipped (eve) gene, is located close to Hoxd-13 and is expressed in limbs like the neighbouring Hoxd genes. To investigate whether this tight linkage reflects a functional similarity, we produced a null allele of Evx-2. Furthermore, and because Hoxd-13 function is prevalent over that of nearby Hoxd genes, we generated two different double mutant loci wherein both Evx-2 and Hoxd-13 were inactivated in cis. The analysis of these various genetic configurations revealed the important function of Evx-2 during the development of the autopod as well as its genetic interaction with Hoxd-13. These results show that, in limbs, Evx-2 functions like a Hoxd gene. A potential evolutionary scenario is discussed, in which Evx-2 was recruited by the HoxD complex in conjunction with the emergence of digits in an ancestral tetrapod

Function of the Evx-2 gene in the morphogenesis of vertebrate limbs.

Vertebrate gene members of the HoxD complex are essential for proper development of the appendicular skeletons. Inactivation of these genes induces severe alterations in the size and number of bony elements. Evx-2, a gene related to the Drosophila even-skipped (eve) gene, is located close to Hoxd-13 and is expressed in limbs like the neighbouring Hoxd genes. To investigate whether this tight linkage reflects a functional similarity, we produced a null allele of Evx-2. Furthermore, and because Hoxd-13 function is prevalent over that of nearby Hoxd genes, we generated two different double mutant loci wherein both Evx-2 and Hoxd-13 were inactivated in cis. The analysis of these various genetic configurations revealed the important function of Evx-2 during the development of the autopod as well as its genetic interaction with Hoxd-13. These results show that, in limbs, Evx-2 functions like a Hoxd gene. A potential evolutionary scenario is discussed, in which Evx-2 was recruited by the HoxD complex in conjunction with the emergence of digits in an ancestral tetrapod

Lipocalin2 Expressions Correlate Significantly With Tumor Differentiation in Epithelial Ovarian Cancer

We recently identified lipocalin2 (LCN2) as being upregulated in ovarian cancer cell lines. The purpose of this study was to validate LCN2 upregulation in ovarian cancers and to investigate its potential as a serum biomarker. We assayed LCN2 expression in ovarian cancers using real-time PCR and IHC. To evaluate the potential of LCN2 as a biomarker, we measured serum LCN2 levels in 54 ovarian cancers, 15 borderline and 53 benign ovarian tumors, and 90 healthy controls. SYBR green PCR and IHC showed LCN2 overexpression in ovarian cancers. LCN2 immunoreactivity was significantly associated with tumor differentiation (p=0.009), as well-differentiated tumors showed the highest LCN2 expression. Serum LCN2 level in ovarian cancer was significantly higher than in the other study groups (p<0.001), and in accordance with IHC results, it also correlated with tumor differentiation, with well-differentiated tumors having the highest value. The sensitivity and specificity of LCN2 in detecting ovarian cancer was 72.2% and 50.4%, respectively. By Cox univariate analysis, LCN2 positivity was an independent prognostic factor for overall survival (hazard ratio = 1.47, p=0.012). In conclusion, LCN2 expressions are upregulated and related to tumor differentiation in ovarian cancers and should be included in future research assessing potential biomarkers for ovarian cancer. (J Histochem Cytochem 57:513–521, 2009