Blood tumor mutational burden and response to pembrolizumab plus chemotherapy in non-small cell lung cancer: KEYNOTE-782.

BACKGROUND: First-line pembrolizumab plus chemotherapy has shown clinical benefit in patients with metastatic non-small cell lung cancer (NSCLC) regardless of tissue tumor mutational burden (tTMB) status. Blood tumor mutational burden (bTMB), assessed using plasma-derived circulating tumor DNA (ctDNA), may be a surrogate for tTMB. The KEYNOTE-782 study evaluated the correlation of bTMB with the efficacy of first-line pembrolizumab plus chemotherapy in NSCLC. METHODS: Previously untreated patients with stage IV nonsquamous NSCLC received pembrolizumab 200 mg plus pemetrexed 500 mg/m2 and investigator's choice of carboplatin area under the curve 5 mg/mL/min or cisplatin 75 mg/m2 for 4 cycles, then pembrolizumab plus pemetrexed for ≤31 additional cycles every 3 weeks. Study objectives were to evaluate the association of baseline bTMB with objective response rate (ORR) (RECIST v1.1 by investigator assessment; primary), progression-free survival (PFS; RECIST v1.1 by investigator assessment), overall survival (OS), and adverse events (AEs; all secondary). A next-generation sequencing assay (GRAIL LLC) with a ctDNA panel that included lung cancer-associated and immune gene targets was used to measure bTMB. RESULTS: 117 patients were enrolled; median time from first dose to data cutoff was 19.3 months (range, 1.0-35.5). ORR was 40.2 % (95 % CI 31.2-49.6 %), median PFS was 7.2 months (95 % CI 5.6-9.8) and median OS was 18.1 months (95 % CI 13.5-25.6). Treatment-related AEs occurred in 113 patients (96.6 %; grade 3-5, n = 56 [47.9 %]). Of patients with evaluable bTMB (n = 101), the area under the receiver operating characteristics curve for continuous bTMB to discriminate response was 0.47 (95 % CI 0.36-0.59). Baseline bTMB was not associated with PFS or OS (posterior probabilities of positive association: 16.8 % and 7.8 %, respectively). CONCLUSIONS: AEs were consistent with the established safety profile of first-line pembrolizumab plus chemotherapy in NSCLC. Baseline bTMB did not show evidence of an association with efficacy.

Analytical validation of a multi-cancer early detection test with cancer signal origin using a cell-free DNA-based targeted methylation assay.

The analytical validation is reported for a targeted methylation-based cell-free DNA multi-cancer early detection test designed to detect cancer and predict the cancer signal origin (tissue of origin). A machine-learning classifier was used to analyze the methylation patterns of >105 genomic targets covering >1 million methylation sites. Analytical sensitivity (limit of detection [95% probability]) was characterized with respect to tumor content by expected variant allele frequency and was determined to be 0.07%-0.17% across five tumor cases and 0.51% for the lymphoid neoplasm case. Test specificity was 99.3% (95% confidence interval, 98.6-99.7%). In the reproducibility and repeatability study, results were consistent in 31/34 (91.2%) pairs with cancer and 17/17 (100%) pairs without cancer; between runs, results were concordant for 129/133 (97.0%) cancer and 37/37 (100%) non-cancer sample pairs. Across 3- to 100-ng input levels of cell-free DNA, cancer was detected in 157/182 (86.3%) cancer samples but not in any of the 62 non-cancer samples. In input titration tests, cancer signal origin was correctly predicted in all tumor samples detected as cancer. No cross-contamination events were observed. No potential interferent (hemoglobin, bilirubin, triglycerides, genomic DNA) affected performance. The results of this analytical validation study support continued clinical development of a targeted methylation cell-free DNA multi-cancer early detection test.

Evaluation of cell-free DNA approaches for multi-cancer early detection.

In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test.

High-intensity sequencing reveals the sources of plasma circulating cell-free DNA variants.

Accurate identification of tumor-derived somatic variants in plasma circulating cell-free DNA (cfDNA) requires understanding of the various biological compartments contributing to the cfDNA pool. We sought to define the technical feasibility of a high-intensity sequencing assay of cfDNA and matched white blood cell DNA covering a large genomic region (508 genes; 2 megabases; >60,000× raw depth) in a prospective study of 124 patients with metastatic cancer, with contemporaneous matched tumor tissue biopsies, and 47 controls without cancer. The assay displayed high sensitivity and specificity, allowing for de novo detection of tumor-derived mutations and inference of tumor mutational burden, microsatellite instability, mutational signatures and sources of somatic mutations identified in cfDNA. The vast majority of cfDNA mutations (81.6% in controls and 53.2% in patients with cancer) had features consistent with clonal hematopoiesis. This cfDNA sequencing approach revealed that clonal hematopoiesis constitutes a pervasive biological phenomenon, emphasizing the importance of matched cfDNA-white blood cell sequencing for accurate variant interpretation.

Acoustic particle filter with adjustable effective pore size for automated sample preparation.

This article presents analysis and optimization of a microfluidic particle filter that uses acoustic radiation forces to remove particles larger than a selected size by adjusting the driving conditions of the piezoelectric transducer (PZT). Operationally, the acoustic filter concentrates microparticles to the center of the microchannel, minimizing undesirable particle adsorption to the microchannel walls. Finite element models predict the complex two-dimensional acoustic radiation force field perpendicular to the flow direction in microfluidic devices. We compare these results with experimental parametric studies including variations of the PZT driving frequencies and voltages as well as various particle sizes (0.5-5.0 microm in diameter). These results provide insight into the optimal operating conditions and show the efficacy of our device as a filter with an adjustable effective pore size. We demonstrate the separation of Saccharomyces cerevisiae from MS2 bacteriophage using our acoustic device. With optimized design of our microfluidic flow system, we achieved yields of greater than 90% for the MS2 with greater than 80% removal of the S. cerevisiae in this continuous-flow sample preparation device.

Detection of 100 aM fluorophores using a high-sensitivity on-chip CE system and transient isotachophoresis.

We present a highly sensitive capillary electrophoresis (CE) assay that combines transient, single-interface on-chip isotachophoresis (ITP) and a laser-induced confocal fluorescence detection setup. We performed experimental parametric studies to show the effects of microscope objective specifications and intensity of excitation laser on optimization of a high-sensitivity on-chip CE detection system. Using the optimized detection system, single-molecule detection of Alexa Fluor 488 was demonstrated, and signal data were validated with autocorrelation analysis. We also demonstrated a separation and detection of 100 aM fluorophores (Alexa Fluor 488 and bodipy) in a fast assay using a high-sensitivity on-chip CE detection system and an ITP/CE protocol with no manual buffer exchange steps. This is, to the knowledge of the authors, the highest electrophoretic separation sensitivity ever reported.

On-chip millionfold sample stacking using transient isotachophoresis.

We present a simple and robust isotachophoresis (ITP) method that can be integrated with microchip-based capillary electrophoresis (CE) devices to achieve millionfold sample stacking. We performed an experimental parametric study to show the effects of initial sample ion concentration, leading ion concentration, and trailing ion concentration on ITP stacking. We also discuss the usefulness and limitations of a simple one-dimensional nondispersive model and a scaling analysis for dispersion rate. We found that a single-column ITP configuration together with electroosmotic flow suppression and high leading ion concentration provide high-performance ITP and can be integrated readily with CE separation. We demonstrated detection of trace of 100 fM Alexa Fluor 488 (signal-to-noise ratio of 11) with a concentration increase of a factor of 2 x 10(6). Application of our ITP/CE protocol to the stacking and separation of negatively charged fluorescent tracers (Alexa Fluor 488 and bodipy) resulted in a concentration increase of 6.4 x 10(4) and a signal increase of 4.5 x 10(5). The ITP/CE protocol can be performed with a standard microchannel cross design or simple flow control. The method can be implemented with available off-the-shelf chip systems using off-the-shelf voltage control systems and buffer chemistries.

Thousandfold signal increase using field-amplified sample stacking for on-chip electrophoresis.

Field-amplified sample stacking (FASS) leverages conductivity gradients between a volume of injected sample and the background buffer to increase sample concentration. A major challenge in applying FASS to on-chip assays is the initial setup of high-conductivity gradient boundaries in the region of the injected sample volume. We have designed, fabricated, and characterized a novel FASS-capillary electrophoresis (CE) chip design that uses a photoinitiated porous polymer structure to facilitate sample injection and flow control for high-gradient FASS. This polymer structure provides a region of high flow resistance that allows the electromigration of sample ions. We have demonstrated an electropherogram signal increase by a factor of 1100 in electrophoretic separations of fluorescein and Bodipy with, respectively, 2 microM and 1 microM initial concentrations.