Assessment of Resistance Mechanisms and Clinical Implications in Patients With EGFR T790M-Positive Lung Cancer and Acquired Resistance to Osimertinib.

Importance: Osimertinib mesylate is used globally to treat EGFR-mutant non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitor resistance mediated by the EGFR T790M mutation. Acquired resistance to osimertinib is a growing clinical challenge that is poorly understood. Objective: To understand the molecular mechanisms of acquired resistance to osimertinib and their clinical behavior. Design, Setting, and Participants: Patients with advanced NSCLC who received osimertinib for T790M-positive acquired resistance to prior EGFR tyrosine kinase inhibitor were identified from a multi-institutional cohort (n = 143) and a confirmatory trial cohort (NCT01802632) (n = 110). Next-generation sequencing of tumor biopsies after osimertinib resistance was performed. Genotyping of plasma cell-free DNA was studied as an orthogonal approach, including serial plasma samples when available. The study and analysis were finalized on November 9, 2017. Main Outcomes and Measures: Mechanisms of resistance and their association with time to treatment discontinuation on osimertinib. Results: Of the 143 patients evaluated, 41 (28 [68%] women) had tumor next-generation sequencing after acquired resistance to osimertinib. Among 13 patients (32%) with maintained T790M at the time of resistance, EGFR C797S was seen in 9 patients (22%). Among 28 individuals (68%) with loss of T790M, a range of competing resistance mechanisms was detected, including novel mechanisms such as acquired KRAS mutations and targetable gene fusions. Time to treatment discontinuation was shorter in patients with T790M loss (6.1 vs 15.2 months), suggesting emergence of pre-existing resistant clones; this finding was confirmed in a validation cohort of 110 patients with plasma cell-free DNA genotyping performed after osimertinib resistance. In studies of serial plasma levels of mutant EGFR, loss of T790M at resistance was associated with a smaller decrease in levels of the EGFR driver mutation after 1 to 3 weeks of therapy (100% vs 83% decrease; P = .01). Conclusions and Relevance: Acquired resistance to osimertinib mediated by loss of the T790M mutation is associated with early resistance and a range of competing resistance mechanisms. These data provide clinical evidence of the heterogeneity of resistance in advanced NSCLC and a need for clinical trial strategies that can overcome multiple concomitant resistance mechanisms or strategies for preventing such resistance.

False-Positive Plasma Genotyping Due to Clonal Hematopoiesis.

Purpose: Plasma cell-free DNA (cfDNA) genotyping is increasingly used in cancer care, but assay accuracy has been debated. Because most cfDNA is derived from peripheral blood cells (PBC), we hypothesized that nonmalignant mutations harbored by hematopoietic cells (clonal hematopoiesis, CH) could be a cause of false-positive plasma genotyping.Experimental Design: We identified patients with advanced non-small cell lung cancer (NSCLC) with KRAS, JAK2, or TP53 mutations identified in cfDNA. With consent, PBC DNA was tested using droplet digital PCR (ddPCR) or next-generation sequencing (NGS) to test for CH-derived mutations.Results: We first studied plasma ddPCR results from 58 patients with EGFR-mutant NSCLC. Two had KRAS G12X detected in cfDNA, and both were present in PBC, including one where the KRAS mutation was detected serially for 20 months. We then studied 143 plasma NGS results from 122 patients with NSCLC and identified 5 JAK2 V617F mutations derived from PBC. In addition, 108 TP53 mutations were detected in cfDNA; for 33 of the TP53 mutations, PBC and tumor NGS were available for comparison, and 5 were present in PBC but absent in tumor, consistent with CH.Conclusions: We find that most JAK2 mutations, some TP53 mutations, and rare KRAS mutations detected in cfDNA are derived from CH not tumor. Clinicians ordering plasma genotyping must be prepared for the possibility that mutations detected in plasma, particularly in genes mutated in CH, may not represent true tumor genotype. Efforts to use plasma genotyping for cancer detection may need paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy. Clin Cancer Res; 24(18); 4437-43. ©2018 AACRSee related commentary by Bauml and Levy, p. 4352.

Discrimination of Germline EGFR T790M Mutations in Plasma Cell-Free DNA Allows Study of Prevalence Across 31,414 Cancer Patients.

Purpose: Plasma cell-free DNA (cfDNA) analysis is increasingly used clinically for cancer genotyping, but may lead to incidental identification of germline-risk alleles. We studied EGFR T790M mutations in non-small cell lung cancer (NSCLC) toward the aim of discriminating germline and cancer-derived variants within cfDNA.Experimental Design: Patients with EGFR-mutant NSCLC, some with known germline EGFR T790M, underwent plasma genotyping. Separately, deidentified genomic data and buffy coat specimens from a clinical plasma next-generation sequencing (NGS) laboratory were reviewed and tested.Results: In patients with germline T790M mutations, the T790M allelic fraction (AF) in cfDNA approximates 50%, higher than that of EGFR driver mutations. Review of plasma NGS results reveals three groups of variants: a low-AF tumor group, a heterozygous group (∼50% AF), and a homozygous group (∼100% AF). As the EGFR driver mutation AF increases, the distribution of the heterozygous group changes, suggesting increased copy number variation from increased tumor content. Excluding cases with high copy number variation, mutations can be differentiated into somatic variants and incidentally identified germline variants. We then developed a bioinformatic algorithm to distinguish germline and somatic mutations; blinded validation in 21 cases confirmed a 100% positive predictive value for predicting germline T790M. Querying a database of 31,414 patients with plasma NGS, we identified 48 with germline T790M, 43 with nonsquamous NSCLC (P < 0.0001).Conclusions: With appropriate bioinformatics, plasma genotyping can accurately predict the presence of incidentally detected germline risk alleles. This finding in patients indicates a need for genetic counseling and confirmatory germline testing. Clin Cancer Res; 23(23); 7351-9. ©2017 AACR.

Controlled self-assembly of alginate microgels by rapidly binding molecule pairs.

Controlled self-assembly of cell-encapsulating microscale polymeric hydrogels (microgels) could be advantageous in a variety of tissue engineering and regenerative medicine applications. Here, a method of assembly by chemical modification of alginate polymer with binding pair molecules (BPM) was explored. Alginate was modified with several types of BPM, specifically biotin and streptavidin and click chemistry compounds, and fabricated into 25-30 µm microgels using a microfluidic platform. These microgels were demonstrated to self-assemble under physiological conditions. By combining complementary microgels at a high ratio, size-defined assemblages were created, and the effects of BPM type and assembly method on the number of microgels per assemblage and packing density were determined. Furthermore, a magnetic process was developed to separate assemblages from single microgels, and allow formation of multilayer spheroids. Finally, cells were singly encapsulated into alginate microgels and assembled using BPM-modified alginate, suggesting potential applications in regenerative medicine.

Deterministic encapsulation of single cells in thin tunable microgels for niche modelling and therapeutic delivery.

Existing techniques to encapsulate cells into microscale hydrogels generally yield high polymer-to-cell ratios and lack control over the hydrogel's mechanical properties. Here, we report a microfluidic-based method for encapsulating single cells in an approximately six-micrometre layer of alginate that increases the proportion of cell-containing microgels by a factor of ten, with encapsulation efficiencies over 90%. We show that in vitro cell viability was maintained over a three-day period, that the microgels are mechanically tractable, and that, for microscale cell assemblages of encapsulated marrow stromal cells cultured in microwells, osteogenic differentiation of encapsulated cells depends on gel stiffness and cell density. We also show that intravenous injection of singly encapsulated marrow stromal cells into mice delays clearance kinetics and sustains donor-derived soluble factors in vivo. The encapsulation of single cells in tunable hydrogels should find use in a variety of tissue engineering and regenerative medicine applications.