Bioanalytical Methods for Characterization of CAR-T Cellular Kinetics: Comparison of PCR Assays and Matrices.

Recently, multiple chimeric antigen receptor T-cell (CAR-T)-based therapies have been approved for treating hematological malignancies, targeting CD19 and B-cell maturation antigen. Unlike protein or antibody therapies, CAR-T therapies are "living cell" therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T-cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR-T therapy trial. In addition, our studies show that the qPCR-based amplification of transgene levels was well-correlated, independent of DNA sources (either CD3+ T-cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR-T dosing prior to expansion and during long-term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.

Long-term outcomes in patients with normal coronary arteries, nonobstructive, or obstructive coronary artery disease on invasive coronary angiography.

BACKGROUND: Normal or near normal coronary arteries (NNCA) or nonobstructive coronary artery disease (CAD) are commonly found on invasive coronary angiography (ICA). HYPOTHESIS: We aimed to determine long-term outcomes by severity of CAD in a contemporary cohort of patients undergoing ICA for evaluation for ischemic heart disease. METHODS: We assessed a consecutive cohort of 925 patients who underwent non-emergent ICA over 24 months. Cardiac death (CD), nonfatal myocardial infarction (NFMI), late revascularization, and medication use were assessed. RESULTS: Follow-up data was available in 850 patients. Of patients without heart failure, at a median of 6.0 years, there was a significant decrease in survival free from CD or NFMI, and from all cardiac events, for those with obstructive CAD compared with patients with NNCAs or nonobstructive CAD (p < .001 for both). No differences between NNCA and nonobstructive CAD patients in rates of CD or NFMI (2.0% vs. 2.1%/year, p = .58) or all cardiac events (2.4% vs. 2.9%/year, p = .84) were observed. CONCLUSION: Long-term follow-up in a contemporary cohort of consecutive patients undergoing non-emergent ICA for detection of CAD showed no difference in annual rates of CD or NFMI, or total cardiac events, in patients with NNCAs versus those with nonobstructive CAD, whereas patients with obstructive CAD had significantly more events. Event rates were low and similar by gender. Use of aspirin, lipid lowering therapy, and beta-blockers increased in all subgroups after ICA. We speculate this may explain the low incidence of subsequent cardiac events, and similar event rates in patients with NNCA and nonobstructive CAD, even in patients presenting with non-ST-elevation MI.

The Lumbosacral Takeoff Angle Can Be Used to Predict the Postoperative Lumbar Cobb Angle Following Selective Thoracic Fusion in Patients with Adolescent Idiopathic Scoliosis.

BACKGROUND: Selective fusion of double curves in patients with scoliosis is considered to spare fusion levels. In 2011, we studied the lumbosacral takeoff angle, defined as the angle between the center-sacral vertical line and a line through the centra of S1, L5, and L4. The lumbosacral takeoff angle was shown to moderately correlate with the lumbar Cobb angle, and a predictive equation was developed to predict the lumbar Cobb angle after selective fusions. The purposes of the present study were to validate that equation in a separate cohort and to assess differences in outcomes following selective and nonselective fusion. METHODS: Patients with Lenke 1B, 1C, 3B, or 3C curve patterns undergoing fusion (both selective and nonselective) with pedicle screw constructs and a minimum of 2 years of follow-up were included. Selective fusion was defined as a lowest level of fixation cephalad to or at the apex of the lumbar curve. To validate the previously derived equation, we used this data set and analysis of variance to check for differences between the actual and calculated postoperative lumbar Cobb angles. Pearson correlation, multiple linear regression, and t tests were used to explore relationships and differences between the selective and nonselective fusion groups. RESULTS: The mean calculated postoperative lumbar Cobb angle (and standard deviation) (22.35° ± 3.82°) was not significantly different from the actual postoperative lumbar Cobb angle (21.08° ± 7.75°), with an average model error of -1.268° (95% confidence interval, -2.649° to 0.112°). The preoperative lumbar Cobb angle was larger in patients with deformities that were chosen for nonselective fusion (50.2° versus 38.9°; p < 0.001). Performing selective fusion resulted in a 3.5° correction of the lumbosacral takeoff angle (p < 0.001), whereas nonselective fusion resulted in a 9.3° correction (p < 0.001). CONCLUSIONS: The lumbosacral takeoff angle can be used to predict the residual lumbar Cobb angle and may be used by surgeons to aid in the decision between selective and nonselective fusion. The change in the lumbosacral takeoff angle following selective fusion is small. Improvement in the lumbosacral takeoff angle and coronal balance is greater in association with nonselective fusion. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.