Use of Advanced Flexible Modeling Approaches for Survival Extrapolation from Early Follow-up Data in two Nivolumab Trials in Advanced NSCLC with Extended Follow-up.

OBJECTIVES: Immuno-oncology (IO) therapies are often associated with delayed responses that are deep and durable, manifesting as long-term survival benefits in patients with metastatic cancer. Complex hazard functions arising from IO treatments may limit the accuracy of extrapolations from standard parametric models (SPMs). We evaluated the ability of flexible parametric models (FPMs) to improve survival extrapolations using data from 2 trials involving patients with non-small-cell lung cancer (NSCLC). METHODS: Our analyses used consecutive database locks (DBLs) at 2-, 3-, and 5-y minimum follow-up from trials evaluating nivolumab versus docetaxel in patients with pretreated metastatic squamous (CheckMate-017) and nonsquamous (CheckMate-057) NSCLC. For each DBL, SPMs, as well as 3 FPMs-landmark response models (LRMs), mixture cure models (MCMs), and Bayesian multiparameter evidence synthesis (B-MPES)-were estimated on nivolumab overall survival (OS). The performance of each parametric model was assessed by comparing milestone restricted mean survival times (RMSTs) and survival probabilities with results obtained from externally validated SPMs. RESULTS: For the 2- and 3-y DBLs of both trials, all models tended to underestimate 5-y OS. Predictions from nonvalidated SPMs fitted to the 2-y DBLs were highly unreliable, whereas extrapolations from FPMs were much more consistent between models fitted to successive DBLs. For CheckMate-017, in which an apparent survival plateau emerges in the 3-y DBL, MCMs fitted to this DBL estimated 5-y OS most accurately (11.6% v. 12.3% observed), and long-term predictions were similar to those from the 5-y validated SPM (20-y RMST: 30.2 v. 30.5 mo). For CheckMate-057, where there is no clear evidence of a survival plateau in the early DBLs, only B-MPES was able to accurately predict 5-y OS (14.1% v. 14.0% observed [3-y DBL]). CONCLUSIONS: We demonstrate that the use of FPMs for modeling OS in NSCLC patients from early follow-up data can yield accurate estimates for RMST observed with longer follow-up and provide similar long-term extrapolations to externally validated SPMs based on later data cuts. B-MPES generated reasonable predictions even when fitted to the 2-y DBLs of the studies, whereas MCMs were more reliant on longer-term data to estimate a plateau and therefore performed better from 3 y. Generally, LRM extrapolations were less reliable than those from alternative FPMs and validated SPMs but remained superior to nonvalidated SPMs. Our work demonstrates the potential benefits of using advanced parametric models that incorporate external data sources, such as B-MPES and MCMs, to allow for accurate evaluation of treatment clinical and cost-effectiveness from trial data with limited follow-up. HIGHLIGHTS: Flexible advanced parametric modeling methods can provide improved survival extrapolations for immuno-oncology cost-effectiveness in health technology assessments from early clinical trial data that better anticipate extended follow-up.Advantages include leveraging additional observable trial data, the systematic integration of external data, and more detailed modeling of underlying processes.Bayesian multiparameter evidence synthesis performed particularly well, with well-matched external data.Mixture cure models also performed well but may require relatively longer follow-up to identify an emergent plateau, depending on the specific setting.Landmark response models offered marginal benefits in this scenario and may require greater numbers in each response group and/or increased follow-up to support improved extrapolation within each subgroup.

Genome-wide profiling of methylation identifies novel targets with aberrant hypermethylation and reduced expression in low-risk myelodysplastic syndromes.

Gene expression profiling signatures may be used to classify the subtypes of Myelodysplastic syndrome (MDS) patients. However, there are few reports on the global methylation status in MDS. The integration of genome-wide epigenetic regulatory marks with gene expression levels would provide additional information regarding the biological differences between MDS and healthy controls. Gene expression and methylation status were measured using high-density microarrays. A total of 552 differentially methylated CpG loci were identified as being present in low-risk MDS; hypermethylated genes were more frequent than hypomethylated genes. In addition, mRNA expression profiling identified 1005 genes that significantly differed between low-risk MDS and the control group. Integrative analysis of the epigenetic and expression profiles revealed that 66.7% of the hypermethylated genes were underexpressed in low-risk MDS cases. Gene network analysis revealed molecular mechanisms associated with the low-risk MDS group, including altered apoptosis pathways. The two key apoptotic genes BCL2 and ETS1 were identified as silenced genes. In addition, the immune response and micro RNA biogenesis were affected by the hypermethylation and underexpression of IL27RA and DICER1. Our integrative analysis revealed that aberrant epigenetic regulation is a hallmark of low-risk MDS patients and could have a central role in these diseases.

High-mobility group and other nonhistone substrates for nuclear histone N-acetyltransferase.

A variety of nonhistone proteins and polyamines has been studied for their substrate activity for nuclear histone N-acetyltransferase. Nonhistone chromatin high-mobility group (HMG) proteins are found to be as good a substrate for the enzyme as histones. The enzyme also acetylates spermidine and spermine. However, protamine, bovine serum albumin, and ubiquitin are not substrates. Chymotryptic peptides of histone and HMGs retained about 64% of the substrate activity, but trypsin treatment reduced the substrate activity by more than 85%. Both N-acetyltransferase activities for HMGs and histones are copurified through salt extraction, polyethylene glycol fractionation, and chromatography on DEAE-cellulose, phosphocellulose columns, and a HPLC anionic-exchange column. The highly purified nuclear histone acetyltransferase shows similar optimal pH and ping-pong kinetics for both HMGs and histones. The Km for HMG is 0.25 mg/ml. HMGs are able to accept the acetyl group from isolated acetyl-enzyme intermediate. Denatured gel analysis shows that HMG 1 and HMG 2 are the major proteins acetylated. High salt concentrations, mononucleotides, and DNA, which inhibit histone substrate activity of the enzyme, also inhibit HMG substrate activity. These observations suggest that there is a major nuclear N-acetyltransferase which is responsible for the acetylation of both histones and HMGs and perhaps also of spermine and spermidine. Thus the regulation of the structure and function of chromatin through postsynthetic acetylation can be achieved by a single nuclear N-acetyltransferase.

Regulation of nuclear histone acetyltransferase by nucleic acids, histone.DNA complex, and chromatin.

Nuclear histone acetyltransferase is found to be inhibited by various nucleic acids and components. Of the adenosine phosphates, the order of inhibitory potency is ATP greater than ADP greater than AMP. Among the nucleoside triphosphates, GTP seems to be the best inhibitor, followed by ATP, CTP, and UTP. Deoxymononucleotides have the same order of inhibition potential as their ribonucleotide counterparts, with inhibition constants in the low millimolar range. Oligonucleotides and polynucleotides are much better inhibitors than mononucleotides. The inhibition constants of the DNA molecules are size dependent. Molecules larger than 40 base pairs have inhibition constants less than 18 micrograms/ml, whereas molecules with decreasing numbers of base pairs have increasing magnitudes of inhibition constants. However, acetyltransferase has a lower affinity for free DNA molecules than for DNA.histone complexes as revealed by its interaction with DNA-Sepharose and histone.DNA-Sepharose columns. Furthermore, native chromatin depleted of endogenous histone acetyltransferase activity shows no inhibitory effect on the enzyme. Yet heated chromatin not only loses substrate activity but also becomes an inhibitor for the enzyme. Since unmodified sea urchin sperm chromatin has been shown to be a potent acetyltransferase inhibitor, it seems possible that DNA.histone complexes may be the true inhibitory species and that the conformational states of such complexes may serve as a regulatory mechanism in the control of the enzyme activity.

Effects of substrates on the thermal stability of nuclear histone acetyltransferase.

Calf thymus nuclear histone acetyltransferase was found to have an optimal activity at about 30 degrees C with an energy of activation of 13.2 +/- 0.4 kcal/mol. The enzyme, however, is thermally unstable. At 38, 42, and 46 degrees C, the enzyme was inactivated with rate constants of 0.01, 0.033 and 0.097 min-1, respectively. High salt concentrations and histones accelerated the rate of thermal inactivation. Bovine serum albumin while alone having no effect on the inactivation process, decreased the exacerbation of heat inactivation caused by histones. Acetyl-CoA, on the other hand, protected the enzyme from heat denaturation. The acetyl-enzyme intermediate also underwent heat inactivated but at a much slower rate. The first order rate constant for the process at 42 degrees C decreased from 0.033 min-1 for the native enzyme to 0.013 min-1 for the acetyl-enzyme, corresponding to an increase in Arrhenius energy of inactivation from 55 kcal/mol to 82 kcal/mol. The distinct effects of acetyl-CoA and histones on the thermal stability of the enzyme suggest that interactions of the enzyme with these two substrates are different and thereby result in different enzyme substrate complexes. This observation further supports our previously proposed two-site ping-pong kinetic reaction mechanism which suggests that the two structurally and electrostatically different substrates bind to separate sites on the enzyme.