Molecular determinants of clinical outcomes with pembrolizumab versus paclitaxel in a randomized, open-label, phase III trial in patients with gastroesophageal adenocarcinoma.

BACKGROUND: In the phase III KEYNOTE-061 trial (NCT02370498), pembrolizumab did not significantly improve overall survival versus paclitaxel as second-line therapy for gastric/gastroesophageal junction (GEJ) adenocarcinoma with programmed death-ligand 1 (PD-L1) combined positive score (CPS) ≥1 tumors. The association of tissue tumor mutational burden (tTMB) status and clinical outcomes was determined, including the relationship with CPS and microsatellite instability-high (MSI-H) status. PATIENTS AND METHODS: In patients with whole exome sequencing (WES) data [420/592 (71%); pembrolizumab, 218; paclitaxel, 202], the association of tTMB with objective response rate (ORR; logistic regression), progression-free survival (PFS; Cox proportional hazards regression), and overall survival (OS; Cox proportional hazards regression) were measured using one-sided (pembrolizumab) and two-sided [paclitaxel] P values. tTMB was also evaluated using FoundationOne®CDx [205/592 (35%)]. Prespecified equivalent cut-offs of 175 mut/exome for WES and 10 mut/Mb for FoundationOne®CDx were used. RESULTS: WES-tTMB was significantly associated with ORR, PFS, and OS in pembrolizumab-treated (all P < 0.001) but not paclitaxel-treated patients (all P > 0.6) in univariate analysis. The area under the receiver operating characteristics curve for WES-tTMB and response was 0.68 [95% confidence interval (CI) 0.56-0.81] for pembrolizumab and 0.51 (95% CI 0.39-0.63) for paclitaxel in univariate analysis. There was low correlation between WES-tTMB and CPS in both treatment groups (r ≤ 0.16). WES-tTMB remained significantly associated with all clinical endpoints with pembrolizumab after adjusting for CPS and with PFS and OS after excluding known MSI-H tumors (n = 26). FoundationOne®CDx-tTMB demonstrated a positive association with ORR, PFS, and OS in pembrolizumab-treated patients (all P ≤ 0.003) but not PFS or OS in paclitaxel-treated patients (P > 0.1). CONCLUSION: This exploratory analysis from KEYNOTE-061 is the first to demonstrate a strong association between tTMB and efficacy with pembrolizumab but not paclitaxel in patients with gastric/GEJ adenocarcinoma in a randomized setting. Data further suggest tTMB is a significant and independent predictor beyond PD-L1 status.

Novel genetic regulation of T helper 1 (Th1)/Th2 cytokine production and encephalitogenicity in inbred mouse strains.

Development of T helper cell (Th)1 or Th2 cytokine responses is essential for effector and regulatory functions of T helper cells. We have compared cytokine profiles of myelin basic protein (MBP) Ac1-16 peptide-specific T helper cells from inbred mouse strains expressing identical k haplotype-derived MHC class II molecules B10.A and B10.BR, B10.BR T cell lines (TCL) produced Th1 cytokines (including high levels of TNF-alpha) and induced experimental autoimmune encephalomyelitis after adoptive transfer. In contrast, B10.A TCL produced Th2 cytokines (including low levels of TNF-alpha) and were poorly encephalitogenic. The contributions of the genetic origin of the T cells and the APC were explored. Serial restimulations of the B10.BR TCL with B10.A or (B10.A x B10.BR) F1 splenic antigen presenting cells (APC) during the establishment of TCL markedly reduced both Th1 cytokine production and encephalitogenicity. In addition, a single restimulation with B10. A splenic APC reduced IFN-gamma and TNF-alpha production by established Th1 MBP-specific Ak-restricted B10.BR TCL and by a Th1 KLH-specific, Ek-restricted B10.BR T cell clone. These studies suggest that B10.A and B10.BR APC differ in their ability to stimulate IFN-gamma and TNF-alpha production by mature Th1 cells and also influence their Th1/Th2 commitment in vivo. The nature of the downregulatory activity of B10.A APC on IFN-gamma and TNF-alpha production was explored. 2-hour supernatants from antigen-activated B10.A APC/TCL cultures or from B10.A APC activated by LPS had the same inhibitory effects on IFN-gamma and TNF-alpha production by B10.BR TCL. The downregulatory effects of B10.A APC are independent of TNF-alpha, IL-4, IL-10, IL-12p40, IFN-gamma, IL-13, TGF-beta, and PGE2. Thus, genetic difference(s) between B10.A and B10.BR APC appear(s) to control the production or activity of a novel soluble cytokine regulatory factor that influences Th1/Th2 commitment and controls production of IFN-gamma and TNF-alpha by mature Th1 cells.

Effect of tumor necrosis factor alpha on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process.

Tumor necrosis factor (TNF) alpha is a cytokine that has potent immune regulatory functions. To assess the potential role of this cytokine in the early development of autoimmunity, we investigated the effect of TNF on the development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice, a spontaneous murine model for autoimmune, insulin-dependent type I diabetes. Treatment of newborn female NOD mice with TNF every other day for 3 wk, led to an earlier onset of disease (10 versus 15 wk of age in control mice) and 100% incidence before 20 wk of age (compared to 45% at 20 wk of age in control phosphate-buffered saline treated female mice). In contrast, administration of an anti-TNF monoclonal antibody, TN3.19.12, resulted in complete prevention of IDDM. In vitro proliferation assays demonstrated that mice treated with TNF developed an increased T cell response to a panel of beta cell autoantigens, whereas anti-TNF treatment resulted in unresponsiveness to the autoantigens. In addition, autoantibody responses to the panel of beta cell antigens paralleled the T cell responses. The effects mediated by TNF appear to be highly age dependent. Treatment of animals either from birth or from 2 wk of age had a similar effect. However, if treatment was initiated at 4 wk of age, TNF delayed disease onset. These data suggest that TNF has a critical role in the early development of autoimmunity towards beta-islet cells.

Capillary electrophoresis of abnormal hemoglobins associated with alpha-thalassemias.

Capillary electrophoresis was evaluated for separation of hemoglobin species associated with alpha-thalassemias, and for identification of hemoglobin variants commonly found in the same human populations. Separation of hemoglobins was achieved using capillary isoelectric focusing with chemical mobilization; visible-wavelength absorbance detection was used to identify hemoglobins against a background of nonheme-containing proteins. This technique could easily differentiate hemoglobins Bart's and H (associated with alpha-thalassemias) from hemoglobin variants. Analysis of globin chains derived from intact hemoglobins was performed by free zone capillary electrophoresis under denaturing conditions. This technique was useful for distinguishing Hb Bart's and Hb H, and for confirming the identity of hemoglobin variants.

[The measurement of intra-cellular AOX in recombinant Pichia pastoris].

The activities of intracellular alcohol oxidase(AOX) in recombinant P. pastoris expressing Pro-UK were determined by a self-designed dissolved oxygen measuring equipment. The enzyme vitality and specific enzyme vitality were defined nd the condition for detecting the enzyme vitality was also established. The experimental results showed that with a certain quantity of biomass in a phosphate buffer containing methanol, the consuming rate of dissolved-oxygen reflected the enzyme vitality of intracellular AOX. It was also found that the pH of the buffer could be very freely between 4.7 and 7.4 and the suitable optical dersity of cell concentration at 600 nm was between 0.5 and 2.0. Furthermore, the values of qo2max and Km of AOX versus oxygen consumption, which were 0.409 s-1 and 0.16 respectively, were calculated. It is a simple and sensitive and feasible method for quick measuring of AOX.

[The chemical decomposition of glutamine and its effect on hybridoma cell culture].

The chemical decomposition of glutamine is a first-order reaction. Its reaction constants under storage and culture conditions were determined as 0.0009 h-1 and 0.0032 h-1 respectively. Batch culture of hybridoma cell C50 with different initial contents of glutamine helped to understand its real metabolic characteristic. The results show that when the initial concentration of glutamine is lower than 5 mmol/L, more than 80% is used by cells. And the lower the initial content, the more being used. As the initial glutamine concentration increases, the ratio of its utilization decreases. When it reaches 10 nmol/L, the ratio decreases dramatically.

Identification of an IFN-gamma responsive region in an intron of the invariant chain gene.

The mechanism by which IFN-gamma up-regulates invariant chain mRNA in antigen-presenting cells has been under intensive investigation. This study shows that in murine monocytic cells the transcriptional up-regulation mediated by the invariant chain (Ii) upstream enhancer only accounts for part of the induction of Ii mRNA by IFN-gamma. We have identified an intronic region in the murine Ii gene that contributes to the transcriptional up-regulation by IFN-gamma. The region has S (H), X/X2 and Y boxes similar to those in MHC class II promoters and the Ii upstream enhancer. Mutations at the putative S, X and Y boxes have abolished the ability of the region to mediate Ii up-regulation by IFN-gamma. Consistent with the functions of these boxes, our findings reveal that the up-regulation of Ii transcription by IFN-gamma mediated by the intronic region is dependent on the induction of class II transactivator by IFN-gamma.

[Study on fermentation of long-chain dicarboxylic acid based on enzymology].

In this paper, we studied the effects of the adding time of alkane on the expression of P450 and production of dicarboxylic acid. A novel fermentation process, in which no or a little alkane was added to make the cells growing more quickly during the growth stage, followed by the addition of alkane to induce cytochromes P450 for 6-8 hours, was established. The results showed that the new process was much better than the old process on inductivities of cytochromes P450 and production of dicarboxylic acid. The new process improved nearly 14.56% expression of P450 and 14.15% production of dicarboxylic acid.

The invariant chain gene intronic enhancer shows homology to class II promoter elements.

Coordinate expression of MHC class II proteins and the class II-associated invariant chain (Ii) is important for proper MHC class II functioning in Ag processing and presentation. The coordinate regulation of these genes results, in part, from the sharing of transcriptional regulatory regions between MHC class II and Ii genes; the Ii has previously been shown to have an upstream enhancer closely related to the essential class II promoter elements. We report here the characterization of a second enhancer in the Ii gene, located within the first intron. This intronic enhancer is contained within a 155-bp region, enhances transcription from the Ii minimal promoter, and also contains elements that are homologous to class II promoter elements X1, X2, and Y boxes.