Yuebi Hu

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.

Amplicon-based next-generation sequencing of plasma cell-free DNA for detection of driver and resistance mutations in advanced non-small cell lung cancer

BackgroundnGenomic analysis of plasma cell-free DNA is transforming lung cancer care; however, available assays are limited by cost, turnaround time, and imperfect accuracy. Here, we study amplicon-based plasma next-generation sequencing (NGS), rather than hybrid-capture-based plasma NGS, hypothesizing this would allow sensitive detection and monitoring of driver and resistance mutations in advanced non-small cell lung cancer (NSCLC).nnnPatients and methodsnPlasma samples from patients with NSCLC and a known targetable genotype (EGFR, ALK/ROS1, and other rare genotypes) were collected while on therapy and analyzed blinded to tumor genotype. Plasma NGS was carried out using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes, as well as intronic coverage for detection of ALK/ROS1 fusions. Diagnostic accuracy was compared with plasma droplet digital PCR (ddPCR) and tumor genotype.nnnResultsnA total of 168 specimens from 46 patients were studied. Matched plasma NGS and ddPCR across 120 variants from 80 samples revealed high concordance of allelic fraction (R2u2009=u20090.95). Pretreatment, sensitivity of plasma NGS for the detection of EGFR driver mutations was 100% (30/30), compared with 87% for ddPCR (26/30). A full spectrum of rare driver oncogenic mutations could be detected including sensitive detection of ALK/ROS1 fusions (8/9 detected, 89%). Studying 25 patients positive for EGFR T790M that developed resistance to osimertinib, 15 resistance mechanisms could be detected including tertiary EGFR mutations (C797S, Q791P) and mutations or amplifications of non-EGFR genes, some of which could be detected pretreatment or months before progression.nnnConclusionsnThis blinded analysis demonstrates the ability of amplicon-based plasma NGS to detect a full range of targetable genotypes in NSCLC, including fusion genes, with high accuracy. The ability of plasma NGS to detect a range of preexisting and acquired resistance mechanisms highlights its potential value as an alternative to single mutation digital PCR-based plasma assays for personalizing treatment of TKI resistance in lung cancer.

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 AACR. See related commentary by Bauml and Levy, p. 4352

Abstract 4112: Overgrowth of competing resistance mechanisms, such as an acquired KRAS mutation, underlies a poor prognosis subtype of acquired resistance to osimertinib in T790M-positive NSCLC

Introduction: Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) which is highly active in EGFR-mutant NSCLC with resistance to prior EGFR TKI. Acquired resistance to osimertinib had been observed clinically; an improved understanding of the molecular mechanisms of resistance is needed. Methods/Results: We initially studied an institutional cohort of 86 patients (pts) treated with osimertinib for advanced T790M-positive NSCLC. 50 pts had progressed on therapy, of whom 25 underwent a resistance biopsy and 17 had NGS results available. 6 pts maintained the T790M mutation at resistance, of whom 3 also acquired an EGFR C797S mutation. The remaining 11 pts had loss of T790M, of whom 5 had evidence of a competing resistance mechanism: 2 with histologic transformation to SCLC, one with BRAF V600E, one with an FGFR3-TACC fusion, and one with KRAS Q61K. For the final case, we confirmed the acquired KRAS Q61K on therapy using serial plasma genotyping. Time to treatment failure (TTF) on osimertinib was 3 months median in pts with loss of T790M and 14 months median in pts with maintained T790M. To test the hypothesis that loss of T790M is a poor prognosis subtype of resistance, we analyzed 127 pts treated for T790M-positive NSCLC on the phase I AURA trial of osimertinib. Plasma drawn after progression was submitted for genotyping using droplet digital PCR. 88 pts had a detectable EGFR driver mutation and were eligible for resistance analysis. 45 pts (51%) had detectable T790M at resistance, 17 (19%) of whom also acquired a C797S mutation; the remaining 43 pts (49%) had loss of T790M and no C797S. Median TTF on osimertinib was 6 months in pts with loss of T790M and 11 months in pts with maintained T790M; among pts with TTF Citation Format: Geoffrey R. Oxnard, Yuebi Hu, Philip Tracy, Nora Feeney, Cloud P. Paweletz, Kenneth S. Thress, Pasi A. Janne. Overgrowth of competing resistance mechanisms, such as an acquired KRAS mutation, underlies a poor prognosis subtype of acquired resistance to osimertinib in T790M-positive NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4112. doi:10.1158/1538-7445.AM2017-4112

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 (nu2009=u2009143) and a confirmatory trial cohort (NCT01802632) (nu2009=u2009110). 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; Pu2009=u2009.01). 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.

Autophagy and Tamoxifen Resistance in Breast Cancer.

Resistance totamoxifen in breast cancer patients is a serious therapeutic problem and majorefforts are underway to understand underlying mechanisms. Resistance can be either intrinsic or acquired. We derived matched tamoxifen sensitive and resistant MCF7 sub-clones that allow us to ask what changes occur in a breast cancer cell when it becomes drug resistant. We consider that this approach mimics de novo tamoxifen resistance and we used these sub-clones to study the factors that lead to drug resistance by two different approaches: expression profiling and an ectopic kinase expression screen. These sub-clones were challenged with steroid depleted media, estrogen, and estrogen plus tamoxifen in order to determine the differential gene response in the sensitive vs. resistant cells. We identified 227 genes that were estrogen responsive in both sub-clones but were not blunted in their response to tamoxifen in the resistant cells. We examined a subset of 67 of these genes that showed differences in baseline expression between sensitive and resistant tumors. Using publicly available data from two different clinical studies of women taking tamoxifen, we found that women classified as matching the sensitive pattern had statistically significant longer disease free survival. The tamoxifen response signature was an independent predictor of outcome compared with other clinical variables by multivariate analysis (p=0.05). The hazard ratio (1.9) was among the strongest observed for the study. Signatures like this one may help personalize therapy by planning regimens of adjuvant therapy best suited to each particular patient. In parallel, high-throughput cell-based screens, in which more than 500 human kinases were independently ectopically expressed, identified 31 kinases that conferred drug resistance on sensitive cells. Both expression profiling and an ectopic kinase expression screen led us to HSPB8, a less well known atypical kinase that has been linked to breast cancer. The ectopic expression of HSPB8 in tamoxifen sensitive cells enabled them to grow in the presence of the drug. Moreover, silencing HSPB8 by shRNA in tamoxifen resistant cells led to cell death. Loss of HSPB8 did not cause cell death by apoptosis, but rather by autophagy.Our findings indicate that HSPB8 is a negative regulator of autophagy and may play an important role in tamoxifen resistance. Interestingly, high expression level of HSPB8 predicted an earlier relapse on tamoxifen in breast cancer patients. Tamoxifen itself induced autophagy in sensitive cells but not in resistant ones and tamoxifen resistant cells were sensitive to the induction of autophagy by other drugs.These results may point to an important role for autophagy in the sensitivity to tamoxifen. We anticipate that this kind of analysis will provide insight into the signaling mechanisms related to the action of estrogen and also may facilitate efforts to identify novel targets for therapeutic intervention for ER positive breast tumors. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5135.