Minimal Residual Disease using a Plasma-Only Circulating Tumor DNA Assay to Predict Recurrence of Metastatic Colorectal Cancer Following Curative Intent Treatment.

PURPOSE: Minimal residual disease (MRD) detection identifies patients with colorectal adenocarcinoma (CRC) likely to recur following definitive treatment. We evaluated a plasma only MRD assay to predict recurrence and survival in metastatic CRC patients undergoing curative intent procedures (surgery and/or radiotherapy), with or without (neo)adjuvant chemotherapy. The primary objective of this study was to assess the correlation of post-procedure tumor cfDNA detection status with radiographic disease recurrence (RFS). EXPERIMENTAL DESIGN: Pre- and post-procedure longitudinal samples were collected from 53 patients and analyzed with a multiomic MRD assay detecting circulating tumor DNA (ctDNA) from genomic and epigenomic signals. Pre- and post-procedure ctDNA detection correlated with recurrence-free and overall survival. RESULTS: 230/233 samples from 52 patients were successfully analyzed. At the time of data cutoff, 36 (69.2%) patients recurred with median follow-up of 31 months. 19/42 patients (45.2%) with ctDNA analyzed 3 weeks post-procedure had detectable ctDNA. ctDNA detection 3 weeks post-procedure was associated with shorter median RFS (HR 5.27; 95% CI, 2.31-12.0, p<0.0001) and overall survival (OS) (HR 12.83; 95% CI, 3.6-45.9, p<0.0001). Pre-procedure ctDNA detection status was not associated with RFS but was associated with improved OS (HR 4.65; 95% CI, 1.4-15.2, p=0.0111). Undetectable ctDNA pre-procedure had notable long-term overall survival, >90% 3 years post-procedure. CONCLUSION: In this cohort of oligometastatic CRC, detection of ctDNA pre- or post-procedure was associated with inferior outcomes even after accounting for prognostic clinicopathologic variables. This suggests ctDNA may enhance current risk stratification methods helping evaluate novel treatments and surveillance strategies toward improving patient outcomes.

A Cell-free DNA Blood-Based Test for Colorectal Cancer Screening.

BACKGROUND: Colorectal cancer is the third most diagnosed cancer in adults in the United States. Early detection could prevent more than 90% of colorectal cancer-related deaths, yet more than one third of the screening-eligible population is not up to date with screening despite multiple available tests. A blood-based test has the potential to improve screening adherence, detect colorectal cancer earlier, and reduce colorectal cancer-related mortality. METHODS: We assessed the performance characteristics of a cell-free DNA (cfDNA) blood-based test in a population eligible for colorectal cancer screening. The coprimary outcomes were sensitivity for colorectal cancer and specificity for advanced neoplasia (colorectal cancer or advanced precancerous lesions) relative to screening colonoscopy. The secondary outcome was sensitivity to detect advanced precancerous lesions. RESULTS: The clinical validation cohort included 10,258 persons, 7861 of whom met eligibility criteria and were evaluable. A total of 83.1% of the participants with colorectal cancer detected by colonoscopy had a positive cfDNA test and 16.9% had a negative test, which indicates a sensitivity of the cfDNA test for detection of colorectal cancer of 83.1% (95% confidence interval [CI], 72.2 to 90.3). Sensitivity for stage I, II, or III colorectal cancer was 87.5% (95% CI, 75.3 to 94.1), and sensitivity for advanced precancerous lesions was 13.2% (95% CI, 11.3 to 15.3). A total of 89.6% of the participants without any advanced colorectal neoplasia (colorectal cancer or advanced precancerous lesions) identified on colonoscopy had a negative cfDNA blood-based test, whereas 10.4% had a positive cfDNA blood-based test, which indicates a specificity for any advanced neoplasia of 89.6% (95% CI, 88.8 to 90.3). Specificity for negative colonoscopy (no colorectal cancer, advanced precancerous lesions, or nonadvanced precancerous lesions) was 89.9% (95% CI, 89.0 to 90.7). CONCLUSIONS: In an average-risk screening population, this cfDNA blood-based test had 83% sensitivity for colorectal cancer, 90% specificity for advanced neoplasia, and 13% sensitivity for advanced precancerous lesions. (Funded by Guardant Health; ECLIPSE ClinicalTrials.gov number, NCT04136002.).

ERBB2 (HER2) amplifications and co-occurring KRAS alterations in the circulating cell-free DNA of pancreatic ductal adenocarcinoma patients and response to HER2 inhibition.

Purpose: Despite accumulating data regarding the genomic landscape of pancreatic ductal adenocarcinoma (PDAC), olaparib is the only biomarker-driven FDA-approved targeted therapy with a PDAC-specific approval. Treating ERBB2(HER2)-amplified PDAC with anti-HER2 therapy has been reported with mixed results. Most pancreatic adenocarcinomas have KRAS alterations, which have been shown to be a marker of resistance to HER2-targeted therapies in other malignancies, though the impact of these alterations in pancreatic cancer is unknown. We describe two cases of ERBB2-amplified pancreatic cancer patients treated with anti-HER2 therapy and provide data on the frequency of ERBB2 amplifications and KRAS alterations identified by clinical circulating cell-free DNA testing. Methods: De-identified molecular test results for all patients with pancreatic cancer who received clinical cell-free circulating DNA analysis (Guardant360) between 06/2014 and 01/2018 were analyzed. Cell-free circulating DNA analysis included next-generation sequencing of up to 73 genes, including select small insertion/deletions, copy number amplifications, and fusions. Results: Of 1,791 patients with pancreatic adenocarcinoma, 36 (2.0%) had an ERBB2 amplification, 26 (72.2%) of whom had a KRAS alteration. Treatment data were available for seven patients. Two were treated with anti-HER2 therapy after their cell-free circulating DNA result, with both benefiting from therapy, including one with a durable response to trastuzumab and no KRAS alteration detected until progression. Conclusion: Our case series illustrates that certain patients with ERBB2-amplified pancreatic adenocarcinoma may respond to anti-HER2 therapy and gain several months of prolonged survival. Our data suggests KRAS mutations as a possible mechanism of primary and acquired resistance to anti-HER2 therapy in pancreatic cancer. Additional studies are needed to clarify the role of KRAS in resistance to anti-HER2 therapy.

Prospective Study of Perioperative Circulating Tumor DNA Dynamics in Patients Undergoing Hepatectomy for Colorectal Liver Metastases.

OBJECTIVE: To evaluate the association of perioperative ctDNA dynamics on outcomes after hepatectomy for CLM. SUMMARY BACKGROUND DATA: Prognostication is imprecise for patients undergoing hepatectomy for CLM, and ctDNA is a promising biomarker. However, clinical implications of perioperative ctDNA dynamics are not well established. METHODS: Patients underwent curative-intent hepatectomy after preoperative chemotherapy for CLM (2013-2017) with paired prehepatectomy/postoperative ctDNA analyses via plasma-only assay. Positivity was determined using a proprietary variant classifier. Primary endpoint was recurrence-free survival (RFS). Median follow-up was 55 months. RESULTS: Forty-eight patients were included. ctDNA was detected before and after surgery (ctDNA+/+) in 14 (29%), before but not after surgery (ctDNA+/-) in 19 (40%), and not at all (ctDNA-/-) in 11 (23%). Adverse tissue somatic mutations were detected in TP53 (n = 26; 54%), RAS (n = 23; 48%), SMAD4 (n = 5; 10%), FBXW7 (n = 3; 6%), and BRAF (n = 2; 4%). ctDNA+/+ was associated with worse RFS (median: ctDNA+/+, 6.0 months; ctDNA+/-, not reached; ctDNA-/-, 33.0 months; P = 0.001). Compared to ctDNA+/+, ctDNA+/- was associated with improved RFS [hazard ratio (HR) 0.24 (95% confidence interval (CI) 0.1-0.58)] and overall survival [HR 0.24 (95% CI 0.08-0.74)]. Adverse somatic mutations were not associated with survival. After adjustment for prehepatectomy chemotherapy, synchronous disease, and ≥2 CLM, ctDNA+/- and ctDNA-/- were independently associated with improved RFS compared to ctDNA+/+ (ctDNA+/-: HR 0.21, 95% CI 0.08-0.53; ctDNA-/-: HR 0.21, 95% CI 0.08-0.56). CONCLUSIONS: Perioperative ctDNA dynamics are associated with survival, identify patients with high recurrence risk, and may be used to guide treatment decisions and surveillance after hepatectomy for patients with CLM.

Monitoring of Dynamic Changes and Clonal Evolution in Circulating Tumor DNA From Patients With IDH-Mutated Cholangiocarcinoma Treated With Isocitrate Dehydrogenase Inhibitors.

PURPOSE: IDH mutations occur in about 30% of patients with cholangiocarcinoma. Analysis of mutations in circulating tumor DNA (ctDNA) can be performed by droplet digital polymerase chain reaction (ddPCR). The analysis of ctDNA is a feasible approach to detect IDH mutations. METHODS: We isolated ctDNA from the blood of patients with IDH-mutated advanced cholangiocarcinoma collected at baseline, on therapy, and at progression to isocitrate dehydrogenase (IDH) inhibitors. RESULTS: Of 31 patients with IDH1R132 (n = 26) or IDH2R172 mutations (n = 5) in the tumor, IDH mutations were detected in 84% of ctDNA samples analyzed by ddPCR and in 83% of ctDNA samples analyzed by next-generation sequencing (NGS). Patients with a low variant allele frequency of ctDNA detected by NGS at baseline had a longer median time to treatment failure compared to patients with high variant allele frequency of ctDNA (3.6 v 1.5 months; P = .008). Patients with a decrease in IDH-mutated ctDNA on therapy by ddPCR compared with no change/increase had a trend to a longer median survival (P = .07). Most frequent emergent alterations in ctDNA by NGS at progression were ARID1A (n = 3) and TP53 mutations (n = 3). CONCLUSION: Detection of IDH mutations in ctDNA in patients with advanced cholangiocarcinoma is feasible, and dynamic changes in ctDNA can correspond with the clinical course and clonal evolution.

Clinical Outcomes for Plasma-Based Comprehensive Genomic Profiling Versus Standard-of-Care Tissue Testing in Advanced Non-Small Cell Lung Cancer.

BACKGROUND: Somatic genomic testing is recommended by numerous expert guidelines to inform targeted therapy treatment for patients with advanced nonsquamous non-small cell lung cancer (aNSCLC). The NILE study was a prospective observational study that demonstrated noninferiority of cell-free circulating tumor DNA (cfDNA)-based tumor genotyping compared to tissue-based genotyping to find targetable genomic alterations in patients with newly diagnosed nonsquamous aNSCLC. As the cohort has matured, clinical outcomes data can now be analyzed. METHODS: This prospective, multicenter North American study enrolled patients with previously untreated nonsquamous aNSCLC who had standard of care (SOC) tissue genotyping performed and concurrent comprehensive cfDNA analysis (Guardant360). Patients with targetable genomic alterations, as defined by NCCN guidelines, who were treated with physician's choice of therapy had objective response rates, disease control rate, and time to treatment collected and compared to published outcomes. RESULTS: Among 282 patients, 89 (31.6%) had an actionable biomarker, as defined by NCCN, detected by tissue (21.3%) and/or cfDNA (27.3%) analysis. Sixty-one (68.5%) of these were treated with an FDA-approved targeted therapy guided by somatic genotyping results (EGFR, ALK, ROS1). Thirty-three patients were eligible for clinical response evaluation and demonstrated an objective response rate of 58% and disease control rate of 94%. Twenty-five (76%) and 17 (52%) achieved a durable response > 6 months and 12 months, respectively. The time to treatment (TtT) was significantly faster for cfDNA-informed biomarker detection as compared to tissue genotyping (18 vs. 31 days, respectively; P = .0008). CONCLUSIONS: cfDNA detects guideline-recommended biomarkers at a rate similar to tissue genotyping, and therapeutic outcomes based on plasma-based comprehensive genomic profiling are comparable to published targeted therapy outcomes with tissue profiling, even in community-based centers.

Landscape and Clonal Dominance of Co-occurring Genomic Alterations in Non-Small-Cell Lung Cancer Harboring MET Exon 14 Skipping.

MET exon 14 skipping alterations (METex14) comprise a diverse set of actionable oncogene drivers in non-small-cell lung cancer (NSCLC). Recent studies have established the efficacy of tyrosine kinase inhibitors for this patient population. The landscape of co-occurring genetic alterations in METex14 NSCLC and their potential impact to therapeutic sensitivities has not yet been fully described. MATERIALS AND METHODS: METex14 NSCLC cases were collected from three cohorts: the VISION trial, and data sets from Guardant360 and GenePlus. Clinicopathologic characteristics and METex14 mutation sites were analyzed and compared across data sets. Co-occurring genetic alterations and the clonality relationships to METex14 were evaluated. RESULTS: Of 40,824 NSCLCs, 692 METex14 cases (1.7%) were identified, including 332 in Guardant360, 188 in VISION, and 172 in GenePlus. The demographics and mutation type and/or sites were similar in the Asian versus Western cohorts. MET amplification, which were found to be associated with sensitivity to MET kinase inhibitors, co-occurs in 7.6%-13.8% of cases, whereas kinase domain secondary mutation of MET co-occurs in 5%-6%. When co-occurring with METex14, EGFR mutations were often identified as the dominant clone (78%, 7 of 9), whereas when co-occurring, METex14 (39%, 7 of 18) and KRAS (44%, 8 of 18) had similar rates of clonal dominance. PIK3CA and PTEN mutations were almost always subclones (89%, 16 of 18) to METex14. Moreover, RET-CCDC6 fusion and EGFR mutation were detected following crizotinib treatment in two patients, suggesting novel mechanisms of resistance. CONCLUSION: METex14 mutations frequently co-occur with other potential driver oncogenes with differing patterns of clonal dominance observed among the drivers. This cellular context can provide insights into whether METex14 is acting as a primary oncogenic driver or resistance mechanism and help guide treatment choices.

Structure-based classification predicts drug response in EGFR-mutant NSCLC.

Epidermal growth factor receptor (EGFR) mutations typically occur in exons 18-21 and are established driver mutations in non-small cell lung cancer (NSCLC)1-3. Targeted therapies are approved for patients with 'classical' mutations and a small number of other mutations4-6. However, effective therapies have not been identified for additional EGFR mutations. Furthermore, the frequency and effects of atypical EGFR mutations on drug sensitivity are unknown1,3,7-10. Here we characterize the mutational landscape in 16,715 patients with EGFR-mutant NSCLC, and establish the structure-function relationship of EGFR mutations on drug sensitivity. We found that EGFR mutations can be separated into four distinct subgroups on the basis of sensitivity and structural changes that retrospectively predict patient outcomes following treatment with EGFR inhibitors better than traditional exon-based groups. Together, these data delineate a structure-based approach for defining functional groups of EGFR mutations that can effectively guide treatment and clinical trial choices for patients with EGFR-mutant NSCLC and suggest that a structure-function-based approach may improve the prediction of drug sensitivity to targeted therapies in oncogenes with diverse mutations.

Erlotinib and Onalespib Lactate Focused on EGFR Exon 20 Insertion Non-Small Cell Lung Cancer (NSCLC): A California Cancer Consortium Phase I/II Trial (NCI 9878).

BACKGROUND: Onalespib is a novel heat shock protein 90 inhibitor (HSP90i). Previous preclinical and clinical studies with HSP90i have demonstrated activity in EGFR-mutant non-small cell lung cancer (NSCLC). This study sought to determine the safety and tolerability of onalespib plus erlotinib in EGFR-mutant NSCLC and to evaluate the preliminary efficacy of the combination in epidermal growth factor receptor exon 20 insertion (EGFRex20ins) NSCLC. PATIENTS AND METHODS: Standard 3+3 dose escalation was followed by a phase II expansion in EGFRex20ins. The phase II component targeted a response rate of 25% versus a background rate of 5%. Prospective next-generation sequencing (NGS) of 70 cancer-related genes, including EGFR, via plasma circulating tumor DNA (ctDNA) was performed. Toxicity was graded by Common Terminology Criteria for Adverse Events (CTCAE), version 4, and response was determined by Response Evaluation Criteria in Solid Tumours (RECIST) 1.1. RESULTS: Eleven patients were treated (nine dose escalation, two dose expansion). Two dose-limiting toxicities (DLTs) occurred in dose level (DL) 0 and zero in DL -1 (minus). In 10 EGFRex20ins patients, no responses were observed, median progression-free survival was 5.4 months (95% confidence interval, 0.9-5.7), and the disease control rate (DCR) was 40% (median, 3.5 months). EGFRex20ins was detected in nine of 10 ctDNA samples at baseline; on-treatment ctDNA clearance was not observed. Grade 3 diarrhea was the predominant toxicity in 45% of patients. The recommended phase II dose is DL -1 (minus): erlotinib 150 mg orally every morning and onalespib 120 mg/m2 intravenously on days 1, 8, and 15 every 28 days. CONCLUSION: Overlapping toxicities of erlotinib and onalespib, mainly diarrhea, limited the tolerability of this combination, and limited clinical activity was observed, so the trial was closed early. Plasma EGFRex20ins ctDNA was detected in the majority of patients; failure to clear ctDNA was consistent with lack of tumor response (NCT02535338).

Clinical and Functional Characterization of Atypical KRAS/NRAS Mutations in Metastatic Colorectal Cancer.

PURPOSE: Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist. EXPERIMENTAL DESIGN: We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations. RESULTS: KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24-6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts. CONCLUSIONS: We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.

Minimal Residual Disease Detection using a Plasma-only Circulating Tumor DNA Assay in Patients with Colorectal Cancer.

PURPOSE: Detection of persistent circulating tumor DNA (ctDNA) after curative-intent surgery can identify patients with minimal residual disease (MRD) who will ultimately recur. Most ctDNA MRD assays require tumor sequencing to identify tumor-derived mutations to facilitate ctDNA detection, requiring tumor and blood. We evaluated a plasma-only ctDNA assay integrating genomic and epigenomic cancer signatures to enable tumor-uninformed MRD detection. EXPERIMENTAL DESIGN: A total of 252 prospective serial plasma specimens from 103 patients with colorectal cancer undergoing curative-intent surgery were analyzed and correlated with recurrence. RESULTS: Of 103 patients, 84 [stage I (9.5%), II (23.8%), III (47.6%), IV (19%)] had evaluable plasma drawn after completion of definitive therapy, defined as surgery only (n = 39) or completion of adjuvant therapy (n = 45). In "landmark" plasma drawn 1-month (median, 31.5 days) after definitive therapy and >1 year follow-up, 15 patients had detectable ctDNA, and all 15 recurred [positive predictive value (PPV), 100%; HR, 11.28 (P < 0.0001)]. Of 49 patients without detectable ctDNA at the landmark timepoint, 12 (24.5%) recurred. Landmark recurrence sensitivity and specificity were 55.6% and 100%. Incorporating serial longitudinal and surveillance (drawn within 4 months of recurrence) samples, sensitivity improved to 69% and 91%. Integrating epigenomic signatures increased sensitivity by 25%-36% versus genomic alterations alone. Notably, standard serum carcinoembryonic antigen levels did not predict recurrence [HR, 1.84 (P = 0.18); PPV = 53.9%]. CONCLUSIONS: Plasma-only MRD detection demonstrated favorable sensitivity and specificity for recurrence, comparable with tumor-informed approaches. Integrating analysis of epigenomic and genomic alterations enhanced sensitivity. These findings support the potential clinical utility of plasma-only ctDNA MRD detection.See related commentary by Bent and Kopetz, p. 5449.

Clinical Impact of Presurgery Circulating Tumor DNA after Total Neoadjuvant Treatment in Locally Advanced Rectal Cancer: A Biomarker Study from the GEMCAD 1402 Trial.

PURPOSE: Total neoadjuvant treatment (TNT) is a valid strategy for patients with high-risk locally advanced rectal cancer (LARC). Biomarkers of response to TNT are an unmet clinical need. We aimed to determine the value of circulating tumor DNA (ctDNA) to predict tumor response, recurrence, and survival in patients with LARC treated with TNT. EXPERIMENTAL DESIGN: The GEMCAD 1402 was a phase II randomized, multicentric clinical trial that randomized 180 patients with LARC to modified schedule of fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) +/- aflibercept, followed by chemoradiation and surgery. Plasma samples were collected at baseline and after TNT within 48 hours before surgery (presurgery). An ultrasensitive assay that integrates genomic and epigenomic cancer signatures was used to assess ctDNA status. ctDNA results were correlated with variables of local tumor response in the surgery sample, local/systemic recurrence, and survival. RESULTS: A total of 144 paired plasma samples from 72 patients were included. ctDNA was detectable in 83% of patients at baseline and in 15% following TNT (presurgery). No association was found between ctDNA status and pathologic response. Detectable presurgery ctDNA was significantly associated with systemic recurrence, shorter disease-free survival (HR, 4; P = 0.033), and shorter overall survival (HR, 23; P < 0.0001). CONCLUSIONS: In patients with LARC treated with TNT, presurgery ctDNA detected minimal metastatic disease identifying patients at high risk of distant recurrence and death. This study sets the basis for prospective clinical trials that use liquid biopsy to personalize the therapeutic approach following TNT.

Genotype-Specific Differences in Circulating Tumor DNA Levels in Advanced NSCLC.

INTRODUCTION: Plasma-based circulating tumor DNA (ctDNA) is an established biomarker for molecular profiling with emerging applications in disease monitoring in multiple tumor types, including, NSCLC. However, determinants of ctDNA shedding and correlation with tumor burden are incompletely understood, particularly in advanced-stage disease. METHODS: We retrospectively analyzed ctDNA-based and tissue-based genomic data and imaging from 144 patients with NSCLC. Tumor burden was quantified with computed tomography (CT) and brain magnetic resonance imaging for the overall cohort and 18F-fludeoxyglucose positron emission tomography-CT in a subset of patients. RESULTS: There was a moderate but statistically significant correlation between ctDNA variant allele frequency and multiple imaging measures of tumor burden such as CT volume (rho = 0.34, p ≤ 0.0001) and metabolic tumor volume (rho = 0.36, p = 0.003). This correlation was strongest in KRAS-mutant tumors (rho = 0.56, p ≤ 0.001), followed by TP53 mutants (rho = 0.43, p ≤ 0.0001), and weakest in EGFR-mutated (EGFR+) tumors (rho = 0.24, p = 0.077). EGFR+ tumors with EGFR copy number gain had significantly higher variant allele frequency than EGFR+ without copy number gain (p ≤ 0.00001). In multivariable analysis, TP53 and EGFR mutations, visceral metastasis, and tumor burden were independent predictors of increased ctDNA shedding. CONCLUSIONS: Levels of detectable ctDNA were affected not only by tumor burden but also by tumor genotype. The genotype-specific differences observed may be due to variations in DNA shedding and cellular turnover. These findings have implications for the emerging use of ctDNA in NSCLC disease monitoring and early detection.

Duration of Targeted Therapy in Patients With Advanced Non-small-cell Lung Cancer Identified by Circulating Tumor DNA Analysis.

BACKGROUND: Outcomes of therapy targeting molecular driver alterations detected in advanced non-small-cell lung (NSCLC) using circulating tumor DNA (ctDNA) have not been widely reported in patients who are targeted therapy-naive. PATIENTS AND METHODS: We performed a multicenter retrospective review of patients with unresectable stage IIIB to IV NSCLC who received matched therapy after a targetable driver alteration was identified using a commercial ctDNA assay through usual clinical care. Eligible patients must not have received targeted therapy prior to ctDNA testing (prior chemotherapy or immunotherapy was permitted). Kaplan-Meier analysis was used to estimate the median duration of targeted therapy. Patients still on targeted therapy were censored at last follow-up. RESULTS: Seventy-six patients met inclusion criteria. The median age of diagnosis of NSCLC was 64.5 years (range, 31-87 years), 67% were female, 74% were never-smokers, and 97% had adenocarcinoma histology. Twenty-one (28%) patients received systemic treatment prior to targeted therapy, including chemotherapy (n = 17), immunotherapy (n = 5), and/or a biologic (n = 4). Thirty-three (43%) patients remain on targeted therapy at the time of data analysis. The median time on targeted therapy was similar to what has been reported for tissue-detected oncogenic driver mutations in the targeted therapy-naive setting. CONCLUSIONS: Patients with ctDNA-detected drivers had durable time on targeted therapy. These treatment outcomes data compliment previous studies that have shown enhanced targetable biomarker discovery rates and high tissue concordance of ctDNA testing when incorporated at initial diagnosis of NSCLC. Identification of NSCLC driver mutations using well-validated ctDNA assays can be used for clinical decision-making and targeted therapy assignment.

Molecular Landscape of BRAF-Mutant NSCLC Reveals an Association Between Clonality and Driver Mutations and Identifies Targetable Non-V600 Driver Mutations.

INTRODUCTION: Approximately 4% of NSCLC harbor BRAF mutations, and approximately 50% of these are non-V600 mutations. Treatment of tumors harboring non-V600 mutations is challenging because of functional heterogeneity and lack of knowledge regarding their clinical significance and response to targeted agents. METHODS: We conducted an integrative analysis of BRAF non-V600 mutations using genomic profiles of BRAF-mutant NSCLC from the Guardant360 database. BRAF mutations were categorized by clonality and class (1 and 2: RAS-independent; 3: RAS-dependent). Cell viability assays were performed in Ba/F3 models. Drug screens were performed in NSCLC cell lines. RESULTS: A total of 305 unique BRAF mutations were identified. Missense mutations were most common (276, 90%), and 45% were variants of unknown significance. F468S and N581Y were identified as novel activating mutations. Class 1 to 3 mutations had higher clonality than mutations of unknown class (p < 0.01). Three patients were treated with MEK with or without BRAF inhibitors. Patients harboring G469V and D594G mutations did not respond, whereas a patient with the L597R mutation had a durable response. Trametinib with or without dabrafenib, LXH254, and lifirafenib had more potent inhibition of BRAF non-V600-mutant NSCLC cell lines than other MEK, BRAF, and ERK inhibitors, comparable with the inhibition of BRAF V600E cell line. CONCLUSIONS: In BRAF-mutant NSCLC, clonality is higher in known functional mutations and may allow identification of variants of unknown significance that are more likely to be oncogenic drivers. Our data indicate that certain non-V600 mutations are responsive to MEK and BRAF inhibitors. This integration of genomic profiling and drug sensitivity may guide the treatment for BRAF-mutant NSCLC.

Routine Plasma-Based Genotyping to Comprehensively Detect Germline, Somatic, and Reversion BRCA Mutations among Patients with Advanced Solid Tumors.

PURPOSE: PARP inhibitors (PARPi) are efficacious in multiple cancers harboring germline (and possibly somatic) BRCA1/2 mutations. Acquired reversions can restore BRCA1/2 function, causing resistance to PARPi and/or platinum-based chemotherapy. The optimal method of identifying patients with germline, somatic, and/or reversion mutations in BRCA1/2 has not been established. Next-generation sequencing (NGS) of cell-free DNA (cfDNA) provides a platform to identify these three types of BRCA1/2 mutations. EXPERIMENTAL DESIGN: Patients with advanced breast, ovarian, prostate, or pancreatic cancer were tested using a clinically validated 73-gene cfDNA assay that evaluates single-nucleotide variants and insertion-deletion mutations (indels) in BRCA1/2, and distinguishes somatic/reversion from germline mutations with high accuracy. RESULTS: Among 828 patients, one or more deleterious BRCA1/2 mutations were detected in 60 (7.2%) patients, including germline (n = 42) and somatic (n = 18) mutations. Common coexisting mutations included TP53 (61.6%), MYC (30%), PIK3CA (26.6%), BRAF (15%), and ESR1 (11.5%). Polyclonal reversion mutations (median, 5) were detected in 9 of 42 (21.4%) germline BRCA1/2-mutant patients, the majority (77.7%) of whom had prior PARPi exposure (median duration, 10 months). Serial cfDNA demonstrated emergence of reversion BRCA mutations under therapeutic pressure from initial PARPi exposure, which contributed to subsequent resistance to PARPi and platinum therapy. CONCLUSIONS: cfDNA NGS identified high rates of therapeutically relevant mutations without foreknowledge of germline or tissue-based testing results, including deleterious somatic BRCA1/2 mutations missed by germline testing and reversion mutations that can have important treatment implications. Further research is needed to confirm clinical utility of these findings to guide precision medicine approaches for patients with advanced malignancies.

Co-occurring Alterations in the RAS-MAPK Pathway Limit Response to MET Inhibitor Treatment in MET Exon 14 Skipping Mutation-Positive Lung Cancer.

PURPOSE: Although patients with advanced-stage non-small cell lung cancers (NSCLC) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood. EXPERIMENTAL DESIGN: Targeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC. RESULTS: Prominent co-occurring RAS-MAPK pathway gene alterations (e.g., in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared with EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by cotreatment with crizotinib and the MEK inhibitor trametinib. CONCLUSIONS: Our study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.

Cell-free Circulating Tumor DNA Variant Allele Frequency Associates with Survival in Metastatic Cancer.

PURPOSE: Physicians are expected to assess prognosis both for patient counseling and for determining suitability for clinical trials. Increasingly, cell-free circulating tumor DNA (cfDNA) sequencing is being performed for clinical decision making. We sought to determine whether variant allele frequency (VAF) in cfDNA is associated with prognosis. EXPERIMENTAL DESIGN: We performed a retrospective analysis of 298 patients with metastatic disease who underwent clinical comprehensive cfDNA analysis and assessed association between VAF and overall survival. RESULTS: cfDNA mutations were detected in 240 patients (80.5%). Median overall survival (OS) was 11.5 months. cfDNA mutation detection and number of nonsynonymous mutations (NSM) significantly differed between tumor types, being lowest in appendiceal cancer and highest in colon cancer. Having more than one NSM detected was associated with significantly worse OS (HR = 2.3; P < 0.0001). VAF was classified by quartiles, Q1 lowest, Q4 highest VAF. Higher VAF levels were associated with a significantly worse overall survival (VAF Q3 HR 2.3, P = 0.0069; VAF Q4 HR = 3.8, P < 0.0001) on univariate analysis. On multivariate analysis, VAF Q4, male sex, albumin level <3.5 g/dL, number of nonvisceral metastatic sites >0 and number of prior therapies >4 were independent predictors of worse OS. CONCLUSIONS: Higher levels of cfDNA VAF and a higher number of NSMs were associated with worse OS in patients with metastatic disease. Further study is needed to determine optimal VAF thresholds for clinical decision making and the utility of cfDNA VAF as a prognostic marker in different tumor types.

Clinical correlates of blood-derived circulating tumor DNA in pancreatic cancer.

BACKGROUND: Treatment outcomes for patients with advanced pancreatic ductal adenocarcinoma (PDAC) remain dismal. There are unmet needs for understanding the biologic basis of this malignancy using novel next-generation sequencing technologies. Herein, we investigated the clinical utility of circulating tumor DNA (ctDNA) (the liquid biopsy) in this malignancy. METHODS: ctDNA was analyzed in 112 patients with PDAC (54-73 genes) and tissue DNA in 66 patients (315 genes) (both clinical-grade next-generation sequencing). Number of alterations, %ctDNA, concordance between ctDNA and tissue DNA, and correlation of ctDNA results with survival were assessed. RESULTS: The most common genes altered in ctDNA were TP53 (46% of patients, N = 51) and KRAS (44%, N = 49). Median number of characterized ctDNA alterations per patient was 1 (range, 0-6), but patients with advanced PDAC had significantly higher numbers of ctDNA alterations than those with surgically resectable disease (median, 2 versus 0.5, P = 0.04). Overall, 75% (70/94) of advanced tumors had ≥ 1 ctDNA alteration. Concordance rate between ctDNA and tissue DNA alterations was 61% for TP53 and 52% for KRAS. Concordance for KRAS alterations between ctDNA and tissue DNA from metastatic sites was significantly higher than between ctDNA and primary tumor DNA (72% vs 39%, P = 0.01). Importantly, higher levels of total %ctDNA were an independent prognostic factor for worse survival (hazard ratio, 4.35; 95% confidence interval, 1.85-10.24 [multivariate, P = 0.001]). A patient with three ctDNA alterations affecting the MEK pathway (GNAS, KRAS, and NF1) attained a response to trametinib monotherapy ongoing at 6 months. CONCLUSIONS: Our findings showed that ctDNA often harbored unique alterations some of which may be targetable and that significantly greater numbers of ctDNA alterations occur in advanced versus resectable disease. Furthermore, higher ctDNA levels were a poor prognostic factor for survival.