Analytical validation of a practical molecular assay prognostic of survival in nonsquamous non-small cell lung cancer.

A molecular assay prognostic of survival in resected nonsquamous non-small cell lung cancer designed to meet the need for improved risk stratification in early-stage disease has recently been described. This assay measures the expression levels of 14 genes using RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. The assay underwent blinded clinical validation in 2 large international cohorts involving approximately 1500 patients; the analytical precision and reproducibility of this assay, however, have not yet been reported. For each of the 14 TaqMan quantitative polymerase chain reaction (PCR) primer and probe sets used in the molecular prognostic assay, the linear range, PCR efficiency, limits of blank, limits of quantitation, and quantitative bias were determined using serial dilutions of pooled RNA extracted from FFPE samples. The reproducibility of the entire molecular assay was determined by performing repeat testing of FFPE samples over multiple days. The linear range of individual quantitative TaqMan PCR primer and probe sets was between 2(10)- and 2(15)-fold input RNA. The median C(T) of the quantitative PCR primer and probe sets at 10 ng of input RNA was 24.3; the median efficiency was 91.2%. The median quantitative bias across all quantitative PCR primer and probe sets was 0.75% (range, 0.32% to 1.32%). In repeat testing, the mean SD of the risk score (scaled from 1 to 100) was 2.18, with a mean coefficient of variation of 0.08. The molecular prognostic assay presented in this study demonstrates high precision and reproducibility, validating its clinical utility as a reliable prognostic tool that can contribute to the management of patients with early-stage disease.

The significance of plasma cell infiltrate in acute cellular rejection of liver allografts.

Acute cellular rejections of higher grades of histologic severity are associated with increased risk of graft failure and death after liver transplantation. Plasma cell-rich infiltrates are associated with adverse clinical outcomes in acute renal allograft rejection and in liver allografts without rejection, but there are limited data on plasma cell-rich infiltrates in acute liver allograft rejection. In this study, 59 biopsies of acute liver allograft rejection were confirmed histologically and clinically, independently graded, and the percentage of plasma cells in portal inflammatory infiltrate was objectively assessed using a standardized protocol. Plasma cell infiltrates were observed in 32 (54%) of the specimens, the mean percentage of plasma cells in the infiltrates being 2.97%. Infiltrates with any plasma cells were significantly more common in groups with higher histologic severity of rejection (75% and 100% versus 31% and 48%, P = .006). The mean percentage of plasma cells in the portal infiltrate was also significantly higher in groups with higher histologic severity of rejection (4.95% and 17.82% versus 0.37 and 0.82%, P = .0002). All the biopsies with more than 30% plasma cells in the infiltrate were found to have severe rejection, whereas all with more than 10% plasma cells had either moderate or severe rejection. The association of plasma cell-rich infiltrates with histologic severity of rejection suggests that plasma cell-rich infiltrates could potentially be useful as a marker of severe rejection.

Prevention of autoimmune diabetes in the DRBB rat by CD40/154 blockade.

The autoimmune diabetes of the DRBB rat shares important similarities with autoimmune diabetes in humans. We have tested the ability of CD40/154 blockade using an anti-CD154 antibody (AH.F5) to prevent autoimmune diabetes in DRBB rats. The rats were treated with two intravenous doses/wk of AH.F5 (15mg/kg/dose) starting at 2-6wks of age. RT6.1 T-cell depletion and poly I/C was started at 4wks of age. Control rats developed diabetes within 25 days after start of depletion therapy. Six of 7, 11 of 13, 7 of 12, and 4 of 11 rats treated with AH.F5 did not develop diabetes when treatment was started at 2-3, 4, 5, and 6wks of age, respectively. The rats that did not develop diabetes were maintained for a minimum of 72 days to >150 days following the last dose of AH.F5. Eleven rats maintained for >150 days underwent an additional depletion and 5/11 developed diabetes within 8-19 days following start of depletion.Histological examination indicated that AH.F5 prevented and possibly reversed insulitis. Islets in about 50% of the treated rats remained free of inflammation following a second course of RT 6.1 T-cell depletion after the serum concentration of AH.F5 was negligible. In summary, CD40/154 blockade with AH.F5 prevents development of autoimmune diabetes if treatment is started prior to overt signs of beta cell destruction. The results indicate that the CD40/154 blockade can prevent diabetes by modifying the expansion or effector phase of the autoimmune diabetes.