In depth evaluation of the prognostic and predictive utility of PTEN immunohistochemistry in colorectal carcinomas: performance of three antibodies with emphasis on intracellular and intratumoral heterogeneity.

BACKGROUND: Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) loss of function is frequently detected in advanced colorectal cancer. Its detection is thought to have prognostic significance and it is being considered to predict responsiveness to anti-EGFR therapy. Unfortunately, while immunohistochemical assessment of PTEN expression is widespread, it lacks standardization and the results are hardly comparable across the available publications. METHODS: Retrospectively collected, formalin-fixed and paraffin-embedded colorectal tumor tissue samples from 55 patients were combined into tissue microarray (TMA) blocks. We used three different PTEN antibodies to determine the frequency, intensity and intracellular pattern of PTEN immunohistochemical labeling: Neomarkers, Dako and CellSignaling. We evaluated the aforementioned parameters in selected regions of colorectal cancers and in their lymph node metastases by using three scoring methods that take into consideration both staining frequency and intensity (H1-H3-score). We also evaluated intracellular localization. RESULTS: The Dako and CellSignaling antibodies stained predominantly cytoplasms, while the Neomarkers antibody specifically stained cell nuclei. PTEN H-scores were significantly lower in all tumor areas as compared to the normal colonic mucosa based on staining with the DAKO and CellSignaling antibodies. Intratumoral regional differences or differences between matching tumors and metastases were not detected with any of the antibodies. Neither Dako, neither CellSignaling, nor the Neomarkers antibodies revealed a significant correlation between PTEN expression and pT, Dukes/MAC and clinical stage. KRAS status, histological grade correlated with PTEN H-scores based on staining with the Neomarkers antibody. PTEN H-scores did not correlate with MMR status. PTEN H-scores did not show any correlation with relapse-free survival based on staining with either antibody. CONCLUSIONS: While PTEN expression decreased in colorectal cancer according to two antibodies, neither of the three applied PTEN antibodies could justify significant correlation with clinicopathological data, nor had prognostic value. Thus, we might conclude that immunohistochemical PTEN investigation remains a challenge requiring more standardized evaluation on larger number of cases to clarify its utility as a prognostic and predictive tool in CRC. The standardization of immunohistochemical method is key in the evaluation process, which is further discussed

Mechanisms of TSC-mediated Control of Synapse Assembly and Axon Guidance

Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, we have examined the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development

Complexity in evaluating resistance of barley (Hordeum vulgare L) to 2 pathotypes of BYDV-PAV: allelic variability at the Yd2 gene locus

International audienc

Evidence of RNA recombination in the genome 3'-terminar region of PAV-Iike isolates of barley yellow dwarf virus (BYDV-PAV)

The genome 3'-terminal region of the PAV-serotype of barley yellow dwarf virus (BYDV-PAV) covers 2 subgenomic RNAs (sgRNA2 and sgRNA3). The sgRNA2 is responsible for the expression of the ORF6 (ORF: open reading frame). The sgRNA3 corresponds to the 334-terminal nucleotides and does not carry coding sequences. In a previous study, we compared the nucleotide sequences of the genome 3'-terminal region for 10 BYDV-PAV isolates differing in their geographical origins and biological properties. In the present investigation we show that the sequence homology grouping obtained for the 5' half of this region is different from that of the 3' half. Therefore, some isolates that are grouped in different clusters according to sequence homologies observed for the 5' half may be grouped in the same cluster according to the 3' half. These differences in sequence homology grouping suggest either different pressures of selection or RNA recombination. The hypothesis of RNA recombination between the 5' half of ancestors of some BYDV-PAV isolates and the 3' half of ancestors of other isolates, leading to isolates differing in their grouping according to both halves, is more favourable. This essentially relies on the fact that the 3' half of the genome 3'-terminal region covers the sgRNA3. The sgRNA3 may have some promoters or structures on its 5' terminus. Being easily recognised by the RNA polymerase, these structures may facilitate RNA recombination by strand switching during replication in mixed infection.Recombinaison ARN entre isolats du BYDV-PAV dans la région 3'-terminale de leur génome. La région 3'-terminale du génome du sérotype PA V du virus de la jaunisse nanisante de l'orge couvre 2 ARN subgénomiques (ARNsg2 et ARNsg3). L'ARNsg2 est responsable de l'expression de l'ORF6. L'ARNsg3, correspondant aux 334-derniers nucléotides, ne porte pas de séquences codantes. Dans une première étude, on a comparé les homologies de séquence de la région 3'-terminale du génome entre 10 isolats du BYDV-PAV d'origines géographiques ou de propriétés biologiques différentes (Chalhoub et al, 1994). Dans la présente investigation, on démontre que le regroupement de ces isolats en fonction des comparaisons de séquences portant sur la moitié 5' de cette région, est différent de celui obtenu avec la moitié 3'. Ainsi, certains isolats groupés dans un même ensemble en fonction des homologies de séquences portant sur la moitié 5' de la région 3'-terminale du génome sont groupés dans des ensembles différents en fonction de la moitié 3'. Ces différences dans le regroupement des isolats suggèrent soit des pressions de sélection différentes, soit des recombinaisons ARN. L'hypothèse de recombinaisons ARN entre la moitié 5' de la région 3'-terminale d'un ancêtre de certains isolats et la moitié 3' d'un ancêtre d'autres isolats semble plus appropriée. En effet, la moitié 3' de la région 3'-terminale du génome couvre l'ARNsg3. Cet ARNsg3 pourrait posséder à son extrémité 5' certains promoteurs ou structures qui, en étant mieux reconnus par l'ARN polymérase, favoriseraient les recombinaisons d'ARN par changement de matrice, pendant la réplication virale lors d'infections mixtes

Evidence of RNA recombination in the genome 3'-terminar region of PAV-Iike isolates of barley yellow dwarf virus (BYDV-PAV)

The genome 3'-terminal region of the PAV-serotype of barley yellow dwarf virus (BYDV-PAV) covers 2 subgenomic RNAs (sgRNA2 and sgRNA3). The sgRNA2 is responsible for the expression of the ORF6 (ORF: open reading frame). The sgRNA3 corresponds to the 334-terminal nucleotides and does not carry coding sequences. In a previous study, we compared the nucleotide sequences of the genome 3'-terminal region for 10 BYDV-PAV isolates differing in their geographical origins and biological properties. In the present investigation we show that the sequence homology grouping obtained for the 5' half of this region is different from that of the 3' half. Therefore, some isolates that are grouped in different clusters according to sequence homologies observed for the 5' half may be grouped in the same cluster according to the 3' half. These differences in sequence homology grouping suggest either different pressures of selection or RNA recombination. The hypothesis of RNA recombination between the 5' half of ancestors of some BYDV-PAV isolates and the 3' half of ancestors of other isolates, leading to isolates differing in their grouping according to both halves, is more favourable. This essentially relies on the fact that the 3' half of the genome 3'-terminal region covers the sgRNA3. The sgRNA3 may have some promoters or structures on its 5' terminus. Being easily recognised by the RNA polymerase, these structures may facilitate RNA recombination by strand switching during replication in mixed infection.Recombinaison ARN entre isolats du BYDV-PAV dans la région 3'-terminale de leur génome. La région 3'-terminale du génome du sérotype PA V du virus de la jaunisse nanisante de l'orge couvre 2 ARN subgénomiques (ARNsg2 et ARNsg3). L'ARNsg2 est responsable de l'expression de l'ORF6. L'ARNsg3, correspondant aux 334-derniers nucléotides, ne porte pas de séquences codantes. Dans une première étude, on a comparé les homologies de séquence de la région 3'-terminale du génome entre 10 isolats du BYDV-PAV d'origines géographiques ou de propriétés biologiques différentes (Chalhoub et al, 1994). Dans la présente investigation, on démontre que le regroupement de ces isolats en fonction des comparaisons de séquences portant sur la moitié 5' de cette région, est différent de celui obtenu avec la moitié 3'. Ainsi, certains isolats groupés dans un même ensemble en fonction des homologies de séquences portant sur la moitié 5' de la région 3'-terminale du génome sont groupés dans des ensembles différents en fonction de la moitié 3'. Ces différences dans le regroupement des isolats suggèrent soit des pressions de sélection différentes, soit des recombinaisons ARN. L'hypothèse de recombinaisons ARN entre la moitié 5' de la région 3'-terminale d'un ancêtre de certains isolats et la moitié 3' d'un ancêtre d'autres isolats semble plus appropriée. En effet, la moitié 3' de la région 3'-terminale du génome couvre l'ARNsg3. Cet ARNsg3 pourrait posséder à son extrémité 5' certains promoteurs ou structures qui, en étant mieux reconnus par l'ARN polymérase, favoriseraient les recombinaisons d'ARN par changement de matrice, pendant la réplication virale lors d'infections mixtes