Brief Report: Combination of Osimertinib and Dacomitinib to Mitigate Primary and Acquired Resistance in EGFR-Mutant Lung Adenocarcinomas.

PURPOSE: Primary and acquired resistance to osimertinib remain significant challenges for patients with EGFR-mutant lung cancers. Acquired EGFR alterations such as EGFR T790M or C797S mediate resistance to EGFR tyrosine kinase inhibitors (TKI) and combination therapy with dual EGFR TKIs may prevent or reverse on-target resistance. PATIENTS AND METHODS: We conducted two prospective, phase I/II trials assessing combination osimertinib and dacomitinib to address on-target resistance in the primary and acquired resistance settings. In the initial therapy study, patients received dacomitinib and osimertinib in combination as initial therapy. In the acquired resistance trial, dacomitinib with or without osimertinib was administered to patients with EGFR-mutant lung cancers with disease progression on osimertinib alone and evidence of an acquired EGFR second-site mutation. RESULTS: Cutaneous toxicities occurred in 93% (any grade) of patients and diarrhea in 72% (any grade) with the combination. As initial therapy, the overall response to the combination was 73% [95% confidence interval (CI), 50%-88%]. No acquired secondary alterations in EGFR were observed in any patients at progression. In the acquired resistance setting, the overall response was 14% (95% CI, 1%-58%). CONCLUSIONS: We observed no acquired secondary EGFR alterations with dual inhibition of EGFR as up-front treatment, but this regimen was associated with greater toxicity. The combination was not effective in reversing acquired resistance after development of a second-site acquired EGFR alteration. Our study highlights the need to develop better strategies to address on-target resistance in patients with EGFR-mutant lung cancers.

Tumor volume as a predictor of cell free DNA mutation detection in advanced non-small cell lung cancer.

Background: Cell free DNA (cfDNA) is an exciting biomarker with applications across the cancer care continuum. Determinants of cfDNA shedding dynamics remain an active research area. We performed a detailed analysis of tumor volume and factors associated with detection of cfDNA mutations. Methods: Patients with advanced non-small cell lung cancers (NSCLCs) were prospectively enrolled on a plasma biomarker protocol. Next generation sequencing (NGS) was performed using a validated, bias-corrected, hybrid-capture panel assay of lung cancer-associated genes. Volume of tumor in different subsites and total tumor volume were determined through manual volume delineation using PET/CT and brain magnetic resonance imaging (MRI) imaging. The primary endpoint was detection of cfDNA mutation; secondary endpoints were overall survival (OS) and variant allele frequency (VAF). Results: There were 110 patients included, 78 of whom had at least one mutation detected. Median total tumor volume for the entire cohort, patients with mutation detected, and patients with no mutation detected were 66 mL (range, 2-1,383 mL), 76 mL (range, 5-1,383 mL), and 45 mL (range, 2-460 mL), respectively (P=0.002; mutation detected vs. not). The optimal total tumor volume threshold to predict increased probability of mutation detection was 65 mL (P=0.006). Total tumor volume greater than 65 mL was a significant predictor of mutation detection on multivariate analysis (OR: 4.30, P=0.003). Significant predictors of OS were age (HR: 1.04, P=0.002), detection of cfDNA mutation (HR: 2.11, P=0.024), and presence of bone metastases (HR: 1.66, P=0.047). Conclusions: Total tumor volume greater than 65 mL was associated with cfDNA mutation detection in patients with advanced NSCLC.

Multiplex SuperSelective PCR Assays for the Detection and Quantitation of Rare Somatic Mutations in Liquid Biopsies.

SuperSelective primers, by virtue of their unique design, enable the simultaneous identification and quantitation of inherited reference genes and rare somatic mutations in routine multiplex PCR assays, while virtually eliminating signals from abundant wild-type sequences closely related to the target mutations. These assays are sensitive, specific, rapid, and low cost, and can be performed in widely available spectrofluorometric thermal cyclers. Herein, we provide examples of SuperSelective PCR assays that target eight different somatic EGFR mutations, irrespective of whether they occur in the same codon, occur at separate sites within the same exon, or involve deletions. In addition, we provide examples of SuperSelective PCR assays that detect specific EGFR mutations in circulating tumor DNA present in the plasma of liquid biopsies obtained from patients with non-small-cell lung cancer. The results suggest that multiplex SuperSelective PCR assays may enable the choice, and subsequent modification, of effective targeted therapies for the treatment of an individual's cancer, utilizing frequent noninvasive liquid biopsies.

Clinical utility of next-generation sequencing-based ctDNA testing for common and novel ALK fusions.

OBJECTIVES: Liquid biopsy for plasma circulating tumor DNA (ctDNA) next-generation sequencing (NGS) can detect ALK fusions, though data on clinical utility of this technology in the real world is limited. MATERIALS AND METHODS: Patients with lung cancer without known oncogenic drivers or who had acquired resistance to therapy (n = 736) underwent prospective plasma ctDNA NGS. A subset of this cohort (n = 497) also had tissue NGS. We evaluated ALK fusion detection, turnaround time (TAT), plasma and tissue concordance, matching to therapy, and treatment response. RESULTS: ctDNA identified an ALK fusion in 21 patients (3%) with a variety of breakpoints and fusion partners, including EML4, CLTC, and PON1, a novel ALK fusion partner. TAT for ctDNA NGS was shorter than tissue NGS (10 vs. 20 days; p < 0.001). Among ALK fusions identified by ctDNA, 93% (13/14, 95% CI 66%-99%) were concordant with tissue evaluation. Among ALK fusions detected by tissue NGS, 54% (13/24, 95% CI 33%-74%) were concordant with plasma ctDNA. ctDNA matched patients to ALK-directed therapy with subsequent clinical response, including four patients matched on the basis of ctDNA results alone due to inadequate or delayed tissue testing. Serial ctDNA analysis detected MET amplification (n = 2) and ALK G1202R mutation (n = 2) as mechanisms of acquired resistance to ALK-directed therapy. CONCLUSION: Our findings support a complementary role for ctDNA in detection of ALK fusions and other alterations at diagnosis and therapeutic resistance settings.

Erlotinib and Trametinib in Patients With EGFR-Mutant Lung Adenocarcinoma and Acquired Resistance to a Prior Tyrosine Kinase Inhibitor.

Inhibition of the MEK/ERK pathway is critical for Bcl-2-like protein 11 (BIM)-mediated epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-induced apoptosis, and dysregulation of this pathway may be a mechanism of acquired resistance. Therefore, MEK inhibition with trametinib and an EGFR TKI may resensitize tumors with acquired resistance. Limited targeted therapies are available after progression on EGFR TKIs, and it is in this setting that we completed a phase I/II study of erlotinib and trametinib. METHODS: Patients with metastatic EGFR-mutant lung adenocarcinoma and acquired resistance to an EGFR TKI received combination erlotinib 75 mg and trametinib 1.5 mg daily until progression or unacceptable side effects. The primary objective was objective response rate determined using RECIST version 1.1. RESULTS: Twenty-three patients were accrued; patients had received a median of two lines of prior TKI therapy (61% prior osimertinib), and 48% had acquired EGFR T790M. We confirmed one partial response (1/23, 4%, 95% CI, 0 to 22). The median progression-free survival was 1.8 months, and the median overall survival was 21 months. Diarrhea (87%), acneiform rash (87%), and fatigue (52%) were the most common treatment-related adverse events. Two patients who had tumor shrinkage both harbored a BRAF fusion. CONCLUSION: Addition of trametinib to erlotinib in the acquired resistance setting in an unselected population is not efficacious. Future studies should focus on targeted therapies in molecularly selected populations. Acquired BRAF fusions in patients with EGFR-sensitizing mutations may be a molecular subset where EGFR and MEK combination therapy could be studied further.

Pilot Study of Dacomitinib for Patients With Metastatic EGFR-Mutant Lung Cancers With Disease Progression After Initial Treatment With Osimertinib.

Patients with EGFR-mutant lung cancer have no approved targeted therapies after disease progression on first-line osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI). Preclinical studies suggest that tumors with both EGFR-sensitizing alteration and acquired second-site EGFR resistance alterations after treatment with osimertinib retain sensitivity to second-generation EGFR TKIs. We hypothesized that dacomitinib, a pan-human epidermal growth factor receptor TKI, may be effective in this setting. METHODS: In this phase II study, patients who had progressed on first-line osimertinib were treated with dacomitinib 45 mg orally daily until disease progression or intolerability. The primary end point was objective response rate. RESULTS: We enrolled 12 patients. Two partial responses were documented (17% objective response rate; 95% CI, 5 to 45). The median progression-free survival was 1.8 months (95% CI, 1.6 to not reached). One patient with an original sensitizing EGFR G719A mutation and one patient without molecular testing available had partial responses, whereas 0 of the 3 patients with second-site acquired EGFR resistance mutations (two C797S and one G724S) met the response criteria. The patient with EGFR G719A has an ongoing response at 17 months, which exceeds prior time on osimertinib (11 months). CONCLUSION: In the first trial evaluating a second-generation EGFR TKI after first-line third-generation osimertinib, we found that dacomitinib after disease progression on osimertinib has limited benefit.

Identification of optimal dosing schedules of dacomitinib and osimertinib for a phase I/II trial in advanced EGFR-mutant non-small cell lung cancer.

Despite the clinical success of the third-generation EGFR inhibitor osimertinib as a first-line treatment of EGFR-mutant non-small cell lung cancer (NSCLC), resistance arises due to the acquisition of EGFR second-site mutations and other mechanisms, which necessitates alternative therapies. Dacomitinib, a pan-HER inhibitor, is approved for first-line treatment and results in different acquired EGFR mutations than osimertinib that mediate on-target resistance. A combination of osimertinib and dacomitinib could therefore induce more durable responses by preventing the emergence of resistance. Here we present an integrated computational modeling and experimental approach to identify an optimal dosing schedule for osimertinib and dacomitinib combination therapy. We developed a predictive model that encompasses tumor heterogeneity and inter-subject pharmacokinetic variability to predict tumor evolution under different dosing schedules, parameterized using in vitro dose-response data. This model was validated using cell line data and used to identify an optimal combination dosing schedule. Our schedule was subsequently confirmed tolerable in an ongoing dose-escalation phase I clinical trial (NCT03810807), with some dose modifications, demonstrating that our rational modeling approach can be used to identify appropriate dosing for combination therapy in the clinical setting.

MET Exon 14-altered Lung Cancers and MET Inhibitor Resistance.

PURPOSE: MET tyrosine kinase inhibitors (TKIs) can achieve modest clinical outcomes in MET exon 14-altered lung cancers, likely secondary to primary resistance. Mechanisms of primary resistance remain poorly characterized and comprehensive proteomic analyses have not previously been performed. EXPERIMENTAL DESIGN: We performed hybrid capture-based DNA sequencing, targeted RNA sequencing, cell-free DNA sequencing, selected reaction monitoring mass spectrometry (SRM-MS), and immunohistochemistry on patient samples of MET exon 14-altered lung cancers treated with a MET TKI. Associations between overall response rate (ORR), progression-free survival (PFS), and putative genomic alterations and MET protein expression were evaluated. RESULTS: Seventy-five of 168 MET exon 14-altered lung cancers received a MET TKI. Previously undescribed (zygosity, clonality, whole-genome duplication) and known (copy-number focality, tumor mutational burden, mutation region/type) genomic factors were not associated with ORR/PFS (P > 0.05). In contrast, MET expression was associated with MET TKI benefit. Only cases with detectable MET expression by SRM-MS (N = 15) or immunochemistry (N = 22) responded to MET TKI therapy, and cancers with H-score ≥ 200 had a higher PFS than cancers below this cutoff (10.4 vs. 5.5 months, respectively; HR, 3.87; P = 0.02). CONCLUSIONS: In MET exon 14-altered cancers treated with a MET TKI, a comprehensive analysis of previously unknown and known genomic factors did not identify a genomic mechanism of primary resistance. Instead, MET expression correlated with benefit, suggesting the potential role of interrogating the proteome in addition to the genome in confirmatory prospective trials.

Effect of Osimertinib and Bevacizumab on Progression-Free Survival for Patients With Metastatic EGFR-Mutant Lung Cancers: A Phase 1/2 Single-Group Open-Label Trial.

Importance: The combination of erlotinib and bevacizumab as initial treatment of epidermal growth factor receptor (EGFR [OMIM 131550])-mutant lung cancers improves progression-free survival (PFS) compared with erlotinib alone. Because osimertinib prolongs PFS compared with erlotinib, this trial was designed to study the combination of osimertinib and bevacizumab as first-line treatment. Objectives: To determine the safety and tolerability of osimertinib and bevacizumab combination treatment and assess the 12-month PFS of the combination in patients with metastatic EGFR-mutant lung cancers. Design, Setting, and Particiants: From August 15, 2016, to May 15, 2018, 49 patients with metastatic EGFR-mutant lung cancers were enrolled in this interventional clinical trial, conducted at a single academic cancer center. In the phase 1 portion of the study, a standard 3 + 3 dose de-escalation design was used to determine the maximum tolerated dose of osimertinib and bevacizumab. In the phase 2 portion of the study, patients were treated at the maximum tolerated dose defined in the phase 1 portion. Statistical analysis was performed from August 1 to October 1, 2019. Interventions: All patients received osimertinib, 80 mg daily, and bevacizumab, 15 mg/kg once every 3 weeks. Main Outcomes and Measures: The primary objective of the phase 2 portion of the study was to determine the number of patients receiving the combination of osimertinib and bevacizumab who were progression free at 12 months. Secondary end points included overall response rate, median PFS, overall survival, and definition of the toxic effects of the combination treatment. Results: Among the 49 patients in the study (34 women; median age, 60 years [range, 36-83 years]), PFS at 12 months was 76% (95% CI, 65%-90%). The overall response rate was 80% (95% CI, 67%-91%), and median PFS was 19 months (95% CI, 15-24 months). Of the 6 patients with measurable central nervous system disease, all had a partial or complete central nervous system response. Persistent detection of EGFR-mutant circulating tumor (ct)DNA at 6 weeks was associated with shorter median PFS (clearance at 6 weeks, 16.2 months [95% CI, 13 months to not reached]; and no clearance at 6 weeks, 9.8 months [95% CI, 4 months to not reached]; P = .04) and median overall survival (clearance at 6 weeks, not reached; and no clearance at 6 weeks, 10.1 months [95% CI, 6 months to not reached]; P = .002). Identified mechanisms of resistance included squamous cell transformation (n = 2) pleomorphic transformation (n = 1), and acquired EGFR L718Q (n = 1) and C797S (n = 1) mutations. Conclusions and Relevance: The combination of osimertinib and bevacizumab met the study's prespecified effectiveness end point. Persistent EGFR-mutant circulating tumor DNA at 6 weeks was associated with early progression and shorter survival. A randomized phase 3 study comparing osimertinib and bevacizumab with osimertinib alone is planned. Trial Registration: ClinicalTrials.gov Identifier: NCT02803203.

Lessons learned from routine, targeted assessment of liquid biopsies for EGFR T790M resistance mutation in patients with EGFR mutant lung cancers.

Background: Plasma cfDNA evaluation at acquired resistance to targeted therapies in lung cancer is routine, however, reports of extended clinical application and pitfalls in laboratory practice are still limited. In this study we describe our experience with cfDNA testing using EGFR T790M as a prototype.Methods: Patients with metastatic EGFR-mutant NSCLC patients who underwent plasma EGFR T790M testing at acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI) from January 2016 through August 2017 were identified. Molecular laboratory records were reviewed to assess performance of testing by digital PCR, concordance between plasma and tissue testing, turnaround time (TAT), plasma T790M variant allele frequency (VAF), and its correlations with metastatic sites and clinical outcomes.Results: 177 patients underwent T790M cfDNA testing during this period. Plasma T790M was positive in 32% of patients. The median TAT was shorter for plasma T790M compared to tissue PCR (9 vs. 15 days, p < .0001), and led to osimertinib use in 84% of positive patients. In 52 patients with plasma and tissue T790M evaluation, the concordance was 77%. Plasma T790M VAF did not correlate with time to osimertinib discontinuation (p = .4). Plasma T790M status correlated with a higher number of metastatic sites (4 vs. 3, p < .001) and bone metastases (p = .0002).Conclusion: Plasma EGFR T790M testing had shorter TAT compared to tissue testing, however, it was longer than anticipated. Test sensitivity is higher in patients with osseous metastases and with higher metastatic burden suggesting a more limited role for early detection. T790M VAF was not associated with clinical outcomes.

A Prospective Study of Circulating Tumor DNA to Guide Matched Targeted Therapy in Lung Cancers.

BACKGROUND: Liquid biopsy for plasma circulating tumor DNA (ctDNA) next-generation sequencing (NGS) is commercially available and increasingly adopted in clinical practice despite a paucity of prospective data to support its use. METHODS: Patients with advanced lung cancers who had no known oncogenic driver or developed resistance to current targeted therapy (n = 210) underwent plasma NGS, targeting 21 genes. A subset of patients had concurrent tissue NGS testing using a 468-gene panel (n = 106). Oncogenic driver detection, test turnaround time (TAT), concordance, and treatment response guided by plasma NGS were measured. All statistical tests were two-sided. RESULTS: Somatic mutations were detected in 64.3% (135/210) of patients. ctDNA detection was lower in patients who were on systemic therapy at the time of plasma collection compared with those who were not (30/70, 42.9% vs 105/140, 75.0%; OR = 0.26, 95% CI = 0.1 to 0.5, P < .001). The median TAT of plasma NGS was shorter than tissue NGS (9 vs 20 days; P < .001). Overall concordance, defined as the proportion of patients for whom at least one identical genomic alteration was identified in both tissue and plasma, was 56.6% (60/106, 95% CI = 46.6% to 66.2%). Among patients who tested plasma NGS positive, 89.6% (60/67; 95% CI = 79.7% to 95.7%) were also concordant on tissue NGS and 60.6% (60/99; 95% CI = 50.3% to 70.3%) vice versa. Patients who tested plasma NGS positive for oncogenic drivers had tissue NGS concordance of 96.1% (49/51, 95% CI = 86.5% to 99.5%), and directly led to matched targeted therapy in 21.9% (46/210) with clinical response. CONCLUSIONS: Plasma ctDNA NGS detected a variety of oncogenic drivers with a shorter TAT compared with tissue NGS and matched patients to targeted therapy with clinical response. Positive findings on plasma NGS were highly concordant with tissue NGS and can guide immediate therapy; however, a negative finding in plasma requires further testing. Our findings support the potential incorporation of plasma NGS into practice guidelines.