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Background Approximately 30% of tumor biopsies from patients with advanced-stage lung adenocarcinomas yield insufficient tissue for successful molecular subtyping. We have analyzed the clinical utility of next-generation sequencing (NGS) of cell-free circulating tumor DNA (ctDNA) in patients with inadequate tumor samples for tissue genotyping. Patients and methods We conducted the study in a multi-institutional prospective cohort of clinically unselected patients with advanced-stage lung adenocarcinomas with insufficient tissue for EGFR, ALK or ROS1 genotyping across 12 Spanish institutions (n  =   93). ctDNA NGS was carried out by Guardant Health (Guardant360, Redwood City, CA), using a hybrid-capture-based 73-gene panel. Variants were deemed actionable if they were part of the OncoKB precision oncology knowledge database and classified in four levels of actionability based on their clinical or preclinical evidence for drug response. Results Eighty-three out of 93 patients (89%) had detectable levels of ctDNA. Potentially actionable level 1–4 genomic alterations were detected in 53 cases (57%), of which 13 (14%) had level 1–2A alterations (Food and Drug Administration-approved and standard-care biomarkers according to lung cancer guidelines). Frequencies of each genomic alteration in ctDNA were consistent with those observed in unselected pulmonary adenocarcinomas. The majority of the patients (62%), particularly those with actionable alterations (87%), had more than one pathogenic variant in ctDNA. The median turnaround time to genomic results was 13 days. Twelve patients (13%) received genotype-matched therapies based on ctDNA results, deriving the expected clinical benefit. Patients with co-occurring pathogenic alterations had a significantly shorter median overall survival as compared with patients without co-occurring pathogenic alteration (multivariate hazard ratio = 5.35, P  =   0.01). Conclusion Digital NGS of ctDNA in lung cancers with insufficient tumor samples for tissue sequencing detects actionable variants that frequently co-occur with other potentially clinically relevant genomic alterations, allowing timely initiation of genotype-matched therapies. [ABSTRACT FROM AUTHOR]

Background Colorectal cancer (CRC) has been shown to acquire RAS and EGFR ectodomain mutations as mechanisms of resistance to epidermal growth factor receptor (EGFR) inhibition (anti-EGFR). After anti-EGFR withdrawal, RAS and EGFR mutant clones lack a growth advantage relative to other clones and decay; however, the kinetics of decay remain unclear. We sought to determine the kinetics of acquired RAS/EGFR mutations after discontinuation of anti-EGFR therapy. Patients and methods We present the post-progression circulating tumor DNA (ctDNA) profiles of 135 patients with RAS / BRAF wild-type metastatic CRC treated with anti-EGFR who acquired RAS and/or EGFR mutations during therapy. Our validation cohort consisted of an external dataset of 73 patients with a ctDNA profile suggestive of prior anti-EGFR exposure and serial sampling. A separate retrospective cohort of 80 patients was used to evaluate overall response rate and progression free survival during re-challenge therapies. Results Our analysis showed that RAS and EGFR relative mutant allele frequency decays exponentially (r 2=0.93 for RAS ; r 2=0.94 for EGFR) with a cumulative half-life of 4.4 months. We validated our findings using an external dataset of 73 patients with a ctDNA profile suggestive of prior anti-EGFR exposure and serial sampling, confirming exponential decay with an estimated half-life of 4.3 months. A separate retrospective cohort of 80 patients showed that patients had a higher overall response rate during re-challenge therapies after increasing time intervals, as predicted by our model. Conclusion These results provide scientific support for anti-EGFR re-challenge and guide the optimal timing of re-challenge initiation. [ABSTRACT FROM AUTHOR]

Background: To identify predictive markers for responders in lapatinib-treated patients and to demonstrate molecular changes during lapatinib treatment via cell-free genomics. Patients and methods: We prospectively evaluated the efficacy of combining lapatinib with capecitabine and oxaliplatin as first line neoadjuvant therapy in patients with previously untreated, HER2-overexpressing advanced gastric cancer. A parallel biomarker study was conducted by simultaneously performing immunohistochemistry and next-generation sequencing (NGS) with tumor and blood samples. Results: Complete response was confirmed in 7/32 patients (21.8%), 2 of whom received radical surgery with pathologicconfirmed complete response. Fifteen partial responses (46.8%) were observed, resulting in a 68.6% overall response rate. NGS of the 16 tumor specimens demonstrated that the most common co-occurring copy number alteration was CCNE1 amplification, which was present in 40% of HER2þ tumors. The relationship between CCNE1 amplification and lack of response to HER2-targeted therapy trended toward statistical significance (66.7% of non-responders versus 22.2% of responders harbored CCNE1 amplification; P=0.08). Patients with high level ERBB2 amplification by NGS were more likely to respond to therapy, compared with patients with low level ERBB2 amplification (P=0.02). Analysis of cfDNA showed that detectable ERBB2 copy number amplification in plasma was predictive to the response (100%, response rate) and changes in plasma-detected genomic alterations were associated with lapatinib sensitivity and/or resistance. The follow-up cfDNA genomics at disease progression demonstrated that there are emergences of other genomic aberrations such as MYC, EGFR, FGFR2 and MET amplifications. Conclusions: The present study showed that HER2þGC patients respond differently according to concomitant genomic aberrations beyond ERBB2, high ERBB2 amplification by NGS or cfDNA can be a positive predictor for patient selection, and tumor genomic alterations change significantly during targeted agent therapy. [ABSTRACT FROM AUTHOR]

Background: While deregulation of the cyclin D1-CDK4/6-retinoblastoma pathway is common in hormone receptor positive (HR+) breast cancer, Rb is usually intact in HR+breast cancer, and targeted CDK 4/6 inhibitors that act upstream of Rb, are routinely being utilized in clinical practice. However, factors that can lead to clinical resistance to CDK 4/6 inhibitors are not known. Patients and methods: We identified patients who had pre- and post-genotyping in tissue and peripheral blood samples after receiving CDK 4/6 inhibitors. Genotyping was carried out in tumor tissue or blood collected before start of CDK 4/6 inhibitor and after disease progression on CDK 4/6 inhibitor, covering more than 90% of the coding region in RB1. Results: We identified detectable acquired RB1 mutations in circulating tumor DNA (ctDNA) after exposure to CDK4/6 inhibitor (palbociclib, palbociclib, ribociclib) for 5, 8, and 13 months, respectively, in three patients. The RB1 mutations included substitution in donor splicing site of exon 8 of the RB1 gene in patient #1; substitution in donor splicing site of exon 22 of RB1 gene, exon 19 deletion, exon 3 insertion in patient #2; and RB1 exon 16 H483Y mutation in patient #3. None of these RB1 mutations were present in the pre-CDK 4/6 specimen highlighting these molecular alterations, which lead to functional loss of Rb1, likely emerged under selective pressure from the CDK4/6 inhibitor potentially confering therapeutic resistance. Conclusion: This is the first clinical report to describe the emergence of somatic RB1 mutations after exposure to palbociclib or ribociclib, in patients with metastatic breast cancer. Further research is needed to validate these findings, identify how these mutations temporally emerge under selective pressure of CDK 4/6 inhibitor, and develop rational therapeutic strategies. [ABSTRACT FROM AUTHOR]

Purpose: BRAF V600E mutations are rarely associated with objective responses to the BRAF inhibitor vemurafenib in patients with metastatic colorectal cancer (CRC). Blockade of BRAF V600E by vemurafenib causes feedback upregulation of EGFR, whose signaling activities can be impeded by cetuximab., Methods: One hundred six patients with BRAF V600E -mutated metastatic CRC previously treated with one or two regimens were randomly assigned to irinotecan and cetuximab with or without vemurafenib (960 mg PO twice daily)., Results: Progression-free survival, the primary end point, was improved with the addition of vemurafenib (hazard ratio, 0.50, P = .001). The response rate was 17% versus 4% ( P = .05), with a disease control rate of 65% versus 21% ( P < .001). A decline in circulating tumor DNA BRAF V600E variant allele frequency was seen in 87% versus 0% of patients ( P < .001), with a low incidence of acquired RAS alterations at the time of progression. RNA profiling suggested that treatment benefit did not depend on previously established BRAF subgroups or the consensus molecular subtype., Conclusion: Simultaneous inhibition of EGFR and BRAF combined with irinotecan is effective in BRAF V600E -mutated CRC.

Purpose: Plasma genotyping may identify mutations in potentially "actionable" cancer genes, such as BRCA1/2 , but their clinical significance is not well-defined. We evaluated the characteristics of somatically acquired BRCA1/2 mutations in patients with metastatic breast cancer (MBC)., Experimental Design: Patients with MBC undergoing routine cell-free DNA (cfDNA) next-generation sequencing (73-gene panel) before starting a new therapy were included. Somatic BRCA1/2 mutations were classified as known germline pathogenic mutations or novel variants, and linked to clinicopathologic characteristics. The effect of the PARP inhibitor, olaparib, was assessed in vitro , using cultured circulating tumor cells (CTCs) from a patient with a somatically acquired BRCA1 mutation and a second patient with an acquired BRCA2 mutation., Results: Among 215 patients with MBC, 29 (13.5%) had somatic cfDNA BRCA1/2 mutations [nine (4%) known germline pathogenic and rest (9%) novel variants]. Known germline pathogenic BRCA1/2 mutations were common in younger patients ( P = 0.008), those with triple-negative disease ( P = 0.022), and they were more likely to be protein-truncating alterations and be associated with TP53 mutations. Functional analysis of a CTC culture harboring a somatic BRCA1 mutation demonstrated high sensitivity to PARP inhibition, while another CTC culture harboring a somatic BRCA2 mutation showed no differential sensitivity. Across the entire cohort, APOBEC mutational signatures (COSMIC Signatures 2 and 13) and the "BRCA" mutational signature (COSMIC Signature 3) were present in BRCA1/2- mutant and wild-type cases, demonstrating the high mutational burden associated with advanced MBC., Conclusions: Somatic BRCA1/2 mutations are readily detectable in MBC by cfDNA analysis, and may be present as both known germline pathogenic and novel variants., (©2020 American Association for Cancer Research.)

Purpose: Recent advances in molecular diagnostic technologies have allowed for the evaluation of solid tumor malignancies via noninvasive blood sampling, including circulating tumor DNA (ctDNA) profiling. We sought to characterize the ctDNA genomic alteration landscape in patients with biliary tract cancers (BTCs)., Patients and Methods: From January 2015 to February 2018, 124 patients with BTC at the Mayo Clinic Comprehensive Cancer Center underwent ctDNA testing using a clinically available assay. The majority of samples (n = 122) were tested using the 73-gene panel that includes somatic genomic targets, including complete or critical exon coverage in 30 and 40 genes, respectively, and in some, amplifications, fusions, and indels., Results: A total of 138 samples were included, with approximately 70% of patients having intrahepatic BTC. All patients had locally advanced or metastatic BTC. Samples with one or more alterations, when variants of unknown significance were excluded, numbered 105 (76%). Each sample contained, on average, three alterations with a median allelic fraction of 0.52%. The overall landscape of alterations is summarized in Figures 1 and 2. After excluding variants of unknown significance, therapeutically relevant alterations were observed in 76 patients (55%), including BRAF mutations, ERBB2 amplifications, FGFR2 fusions, FGFR2 mutations, and IDH1 mutations seen in 21% of patients. A different spectrum of alterations was observed in patients with early-onset BTC (younger than age 50 years) compared with older patients (older than age 50 years)., Conclusion: Data on ctDNA in BTC is currently limited. Our study, the largest cohort reported to date to our knowledge, demonstrates the feasibility of ctDNA testing in this disease. We provide a foundation upon which the field can continue to grow.

Purpose: Gene fusions are established oncogenic drivers and emerging therapeutic targets in advanced colorectal cancer. This study aimed to detail the frequencies and clinicopathological features of gene fusions in colorectal cancer using a circulating tumor DNA assay., Methods: Circulating tumor DNA samples in patients with advanced colorectal cancer were analyzed at 4,581 unique time points using a validated plasma-based multigene assay that includes assessment of fusions in FGFR2 , FGFR3 , RET , ALK , NTRK1 , and ROS1. Associations between fusions and clinicopathological features were measured using Fisher's exact test. Relative frequencies of genomic alterations were compared between fusion-present and fusion-absent cases using an unpaired t test., Results: Forty-four unique fusions were identified in 40 (1.1%) of the 3,808 patients with circulating tumor DNA detected: RET (n = 6; 36% of all fusions detected), FGFR3 (n = 2; 27%), ALK (n = 10, 23%), NTRK1 (n = 3; 7%), ROS1 (n = 2; 5%), and FGFR2 (n = 1; 2%). Relative to nonfusion variants detected, fusions were more likely to be subclonal (odds ratio, 8.2; 95% CI, 2.94 to 23.00; P < .001). Mutations associated with a previously reported anti-epidermal growth factor receptor (anti-EGFR) therapy resistance signature (subclonal RAS and EGFR mutations) were found with fusions in FGFR3 (10 of 12 patients), RET (nine of 16 patients), and ALK (seven of 10 patients). For the 27 patients with available clinical histories, 21 (78%) had EGFR monoclonal antibody treatment before fusion detection., Conclusion: Diverse and potentially actionable fusions can be detected using a circulating tumor DNA assay in patients with advanced colorectal cancer. Distribution of coexisting subclonal mutations in EGFR , KRAS , and NRAS in a subset of the patients with fusion-present colorectal cancer suggests that these fusions may arise as a novel mechanism of resistance to anti-EGFR therapies in patients with metastatic colorectal cancer., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Katherine CliftonEmployment: WellCare (I) Stock and Other Ownership Interests: WellCare (I)Thereasa A. RichEmployment: Guardant Health Stock and Other Ownership Interests: Guardant Health, Myriad GeneticsVictoria M. RaymondEmployment: TrovaGene, Guardant Health Stock and Other Ownership Interests: TrovaGene, Guardant HealthArvind DasariConsulting or Advisory Role: Ipsen, AbbVie/Stemcentrx, Novartis, Voluntis, Lexicon Research Funding: Novartis, eFFECTOR Therapeutics, Eisai, Hutchison MediPharma, Merck, Guardant HealthJonathan M. LoreeConsulting or Advisory Role: Taiho Pharma Canada, Ipsen, Novartis, Bayer, AmgenTodd M. BauerEmployment: Tennessee Oncology Consulting or Advisory Role: Ignyta (Inst), Guardant Health, Loxo, Pfizer, Moderna Therapeutics (Inst), Pfizer (Inst), Exelixis Speakers' Bureau: Bayer Research Funding: Daiichi Sankyo (Inst), MedPacto (Inst), Incyte (Inst), Mirati Therapeutics (Inst), MedImmune (Inst), AbbVie (Inst), AstraZeneca (Inst), Leap Therapeutics (Inst), MabVax Therapeutics (Inst), Stemline Therapeutics (Inst), Merck (Inst), Eli Lilly (Inst), GlaxoSmithKline (Inst), Novartis (Inst), Pfizer (Inst), Genentech (Inst), Deciphera Pharmaceuticals (Inst), Merrimack (Inst), Immunogen (Inst), Millennium (Inst), Ignyta (Inst), Calithera Biosciences (Inst), Kolltan Pharmaceutical (Inst), Principia Biopharma (Inst), Peleton (Inst), Immunocore (Inst), Roche (Inst), Aileron Therapeutics (Inst), Bristol-Myers Squibb (Inst), Amgen (Inst), Moderna Therapeutics (Inst), Sanofi (Inst), Boehringer Ingelheim (Inst), Astellas Pharma (Inst), Five Prime Therapeutics (Inst), Jacobio Pharmaceuticals (Inst), Top Alliance BioSciences (Inst), Loxo (Inst), Janssen (Inst), Clovis Oncology (Inst), Takeda (Inst), Karyopharm Therapeutics (Inst), Onyx (Inst), Phosplatin Therapeutics (Inst), Foundation Medicine (Inst), ARMO BioSciences (Inst) Travel, Accommodations, Expenses: Astellas Pharma, AstraZeneca, Celgene, Clovis Oncology, EMD Serono, Genentech, Eli Lilly, Merck, Novartis, Pharmacyclics, SysmexYoung Kwang ChaeConsulting or Advisory Role: Foundation Medicine, Boehringer Ingelheim, Biodesix, Counsyl, AstraZeneca, Guardant Health, Takeda, Genentech, ImmuneOncia, Hanmi Speakers' Bureau: Genentech, Merck, AstraZeneca, Eli Lilly Research Funding: AbbVie, Bristol-Myers Squibb, Lexent Bio, Freenome, Biodesix Travel, Accommodations, Expenses: HanmiAxel GrotheyHonoraria: Elsevier, Aptitude Health, Imedex Consulting or Advisory Role: Genentech (Inst), Bayer (Inst), Bristol-Myers Squibb (Inst), Eli Lilly (Inst), Boston Biomedical (Inst), Amgen (Inst), Array BioPharma (Inst), Guardant Health (Inst), Daiichi Sankyo (Inst) Research Funding, Genentech (Inst), Bayer (Inst), Pfizer (Inst), Eisai (Inst), Eli Lilly (Inst), Boston Biomedical (Inst), Daiichi Sankyo (Inst), Array BioPharma (Inst) Travel, Accommodations, Expenses: Genentech, Bayer, Bristol-Myers Squibb, Boston Biomedical, Amgen, Boehringer Ingelheim, Merck Sharp & DohmeAndrew HendifarConsulting or Advisory Role: Novartis, Ipsen, Perthera, Celgene, AbbVie Research Funding: Ipsen Travel, Accommodations, Expenses: HalozymeDavid HenryHonoraria: Amgen, AMAG Pharmaceuticals Consulting or Advisory Role: AMAG Pharmaceuticals, Amgen Research Funding: Amgen Travel, Accommodations, Expenses: Amgen, AMAG PharmaceuticalsDaruka MahadevanHonoraria: Guardant Health, Caris Life Sciences Speakers' Bureau: Guardant Health, Caris Life Sciences Travel, Accommodations, Expenses: Guardant Health, Caris Life SciencesMohammad NezamiEmployment: Orange Coast Medical Center of Hope Stock and Other Ownership Interests: Sahel OncologyBenjamin TanConsulting or Advisory Role: Genentech Research Funding: Genentech, Eli Lilly, Pfizer, Bayer, Eisai, Exelixis, Merck Serono, Tyrogenex, SillaJen, Boehringer IngelheimZev A. WainbergConsulting or Advisory Role: Array BioPharma, Five Prime Therapeutics, Novartis, Eli Lilly, Merck, Merck KGaA, Bristol-Myers Squibb, Genentech, Bayer Research Funding: Novartis (Inst), Plexxikon (Inst), Pfizer (Inst), Merck (Inst), Five Prime Therapeutics (Inst) Travel, Accommodations, Expenses: GenentechRichard LanmanEmployment: Guardant Health, Veracyte Leadership: Guardant Health, Biolase Stock and Other Ownership Interests: Guardant Health, Biolase, Forward Medical Research Funding: Guardant HealthScott KopetzStock and Other Ownership Interests: MolecularMatch, Navire Consulting or Advisory Role: Roche, Genentech, EMD Serono, Merck, Karyopharm Therapeutics, Amal Therapeutics, Navire Pharma, Symphogen, Holy Stone, Biocartis, Amgen, Novartis, Eli Lilly, Boehringer Ingelheim, Boston Biomedical, AstraZeneca/MedImmune, Bayer Health, Pierre Fabre, EMD Serono Research Funding: Amgen (Inst), Sanofi (Inst), Biocartis (Inst), Guardant Health (Inst), Array BioPharma (Inst), Genentech (Inst), EMD Serono (Inst), MedImmune (Inst), Novartis (Inst), Eli Lilly (Inst)Van MorrisConsulting or Advisory Role: Clinical Genomics, Array Biopharma Research Funding: Bristol-Myers Squibb (Inst), Array BioPharma (Inst), EMD Serono (Inst) Travel, Accommodations, Expenses: Array BioPharma No other potential conflicts of interest were reported., (© 2019 by American Society of Clinical Oncology.)

Purpose: We describe herein a novel P447&#95;L455 deletion in the C2 domain of PIK3CA in a patient with an ER + breast cancer with an excellent response to the PI3Kα inhibitor alpelisib. Although PIK3CA deletions are relatively rare, a significant portion of deletions cluster within amino acids 446-460 of the C2 domain, suggesting these residues are critical for p110α function. Experimental Design: A computational structural model of PIK3CA delP447-L455 in complex with the p85 regulatory subunit and MCF10A cells expressing PIK3CA delP447-L455 and PIK3CA H450&#95;P458del were used to understand the phenotype of C2 domain deletions. Results: Computational modeling revealed specific favorable inter-residue contacts that would be lost as a result of the deletion, predicting a significant decrease in binding energy. Coimmunoprecipitation experiments showed reduced binding of the C2 deletion mutants with p85 compared with wild-type p110α. The MCF10A cells expressing PIK3CA C2 deletions exhibited growth factor-independent growth, an invasive phenotype, and higher phosphorylation of AKT, ERK, and S6 compared with parental MCF10A cells. All these changes were ablated by alpelisib treatment. Conclusions: C2 domain deletions in PIK3CA generate PI3K dependence and should be considered biomarkers of sensitivity to PI3K inhibitors. Clin Cancer Res; 24(6); 1426-35. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Gastroesophageal adenocarcinoma (GEA) is a lethal disease where targeted therapies, even when guided by genomic biomarkers, have had limited efficacy. A potential reason for the failure of such therapies is that genomic profiling results could commonly differ between the primary and metastatic tumors. To evaluate genomic heterogeneity, we sequenced paired primary GEA and synchronous metastatic lesions across multiple cohorts, finding extensive differences in genomic alterations, including discrepancies in potentially clinically relevant alterations. Multiregion sequencing showed significant discrepancy within the primary tumor (PT) and between the PT and disseminated disease, with oncogene amplification profiles commonly discordant. In addition, a pilot analysis of cell-free DNA (cfDNA) sequencing demonstrated the feasibility of detecting genomic amplifications not detected in PT sampling. Lastly, we profiled paired primary tumors, metastatic tumors, and cfDNA from patients enrolled in the personalized antibodies for GEA (PANGEA) trial of targeted therapies in GEA and found that genomic biomarkers were recurrently discrepant between the PT and untreated metastases. Divergent primary and metastatic tissue profiling led to treatment reassignment in 32% (9/28) of patients. In discordant primary and metastatic lesions, we found 87.5% concordance for targetable alterations in metastatic tissue and cfDNA, suggesting the potential for cfDNA profiling to enhance selection of therapy. Significance: We demonstrate frequent baseline heterogeneity in targetable genomic alterations in GEA, indicating that current tissue sampling practices for biomarker testing do not effectively guide precision medicine in this disease and that routine profiling of metastatic lesions and/or cfDNA should be systematically evaluated. Cancer Discov; 8(1); 37-48. ©2017 AACR. See related commentary by Sundar and Tan, p. 14 See related article by Janjigian et al., p. 49 This article is highlighted in the In This Issue feature, p. 1 ., (©2017 American Association for Cancer Research.)