Cell lines LIM2405, LIM1899 and HT29 were tested in quadruplicate and repeated in three separate PCR assays. The assay was the two precise and reproducible the implies for LIM2405, LIM1899 and HT29 were one. 08 SEM 0. 04, 2. 07 SEM 0. 03 and two. 96 SEM 0. 07 respectively, plus the coefficient of vari ation from run to run was 2. 4%, and intra assay CV was concerning 0. 12% and 0. 99%. These cell lines were there fore used as 1, two and 3 copy controls respectively. Our group has previously described quantification of PTEN gene copy quantity on cell lines LIM2405 and LIM1899. For inhibitor Ridaforolimus the patients DNA, reduction of PTEN was defined as one. five copies, no loss was one. five copies. Benefits Fifty 9 tumor specimens were analysed for loss of copy variety by Taqman and for reduction of protein expres sion by IHC.
Eight samples have been discovered to contain no tumor tissue and have been excluded from even more evaluation. Immunohistochemistry Two blinded pathologists assessed 51 specimens Camostat Mesilate independ ently for PTEN protein expression with IHC. Pathologist JC assessed 29 51 as acquiring PTEN expression reduction, while pathologist AR assessed 17 51 as owning loss of PTEN expression. Concordance amongst pathologists on last IHC evaluation was 37 51, indicating in 14 51 of specimens there was discordance from the final assessment of IHC PTEN loss. Taqman PCR. Seventeen specimens had PTEN allelic loss on Taqman PCR of which 10 had PTEN loss on IHC. Fifteen specimens had preserved PTEN on each IHC and Taqman PCR evaluation. All round concord ance amongst IHC and Taqman copy number in PTEN loss assessment was 25 37.
Discussion On this validation research of PTEN evaluation in CRC we evaluated inter observer variability in PTEN evaluation with IHC and subsequently the discordance of PTEN assessment amongst IHC and PCR based mostly methodologies. IHC evaluation yielded prices of PTEN reduction of 33% and 57% in between two pathologists, whilst Taqman PCR dem onstrated 49% of specimens contained PTEN allelic reduction. Our examination provides distinct insight into the relation ship amongst PTEN protein expression and allelic loss. Particularly how is protein expression maintained during the setting of allelic loss, and why do samples present absence of PTEN expression regardless of allelic reduction In samples with PTEN allelic reduction 41% maintained pro tein expression. Of those specimens all had IHC staining intensity of one suggesting possibly a diminished level of PTEN protein. The upkeep of protein expression in these situations is possible as a consequence of the remaining functional PTEN allele, which makes it possible for transcription of the normal PTEN protein. In cases of PTEN haploinsufficiency no matter if protein expression is diminished Taqman copy number PCR Making use of a PTEN Taqman copy number assay, 25 51 specimens had one. five copy amount and have been consequently classified as PTEN loss.