Circulating Tumor DNA Identifies Diverse Landscape of Acquired Resistance to Anti-Epidermal Growth Factor Receptor Therapy in Metastatic Colorectal Cancer.

PURPOSE: Anti-epidermal growth factor receptor (EGFR) antibodies are effective treatments for metastatic colorectal cancer. Improved understanding of acquired resistance mechanisms may facilitate circulating tumor DNA (ctDNA) monitoring, anti-EGFR rechallenge, and combinatorial strategies to delay resistance. METHODS: Patients with treatment-refractory metastatic colorectal cancer (n = 169) enrolled on the CO.26 trial had pre-anti-EGFR tissue whole-exome sequencing (WES) compared with baseline and week 8 ctDNA assessments with the GuardantOMNI assay. Acquired alterations were compared between patients with prior anti-EGFR therapy (n = 66) and those without. Anti-EGFR therapy occurred a median of 111 days before ctDNA assessment. RESULTS: ctDNA identified 12 genes with increased mutation frequency after anti-EGFR therapy, including EGFR (P = .0007), KRAS (P = .0017), LRP1B (P = .0046), ZNF217 (P = .0086), MAP2K1 (P = .018), PIK3CG (P = .018), BRAF (P = .048), and NRAS (P = .048). Acquired mutations appeared as multiple concurrent subclonal alterations, with most showing decay over time. Significant increases in copy-gain frequency were noted in 29 genes after anti-EGFR exposure, with notable alterations including EGFR (P < .0001), SMO (P < .0001), BRAF (P < .0001), MET (P = .0002), FLT3 (P = .0002), NOTCH4 (P = .0006), ERBB2 (P = .004), and FGFR1 (P = .006). Copy gains appeared stable without decay 8 weeks later. There were 13 gene fusions noted among 11 patients, all but one of which was associated with prior anti-EGFR therapy. Polyclonal resistance was common with acquisition of ≥ 10 resistance related alterations noted in 21% of patients with previous anti-EGFR therapy compared with 5% in those without (P = .010). Although tumor mutation burden (TMB) did not differ pretreatment (P = .63), anti-EGFR exposure increased TMB (P = .028), whereas lack of anti-EGFR exposure resulted in declining TMB (P = .014). CONCLUSION: Paired tissue and ctDNA sequencing identified multiple novel mutations, copy gains, and fusions associated with anti-EGFR therapy that frequently co-occur as subclonal alterations in the same patient.

Identification of KRASG12C Mutations in Circulating Tumor DNA in Patients With Cancer.

PURPOSE: KRAS is the most mutated proto-oncogene that has been identified in cancer, and treatment of patients with KRAS mutations remains an arduous challenge. Recently, KRASG12C mutation has attracted special interest because it is now considered potentially druggable with recently developed covalent small-molecule KRASG12C inhibitors. Nevertheless, to date, there have been no large-scale analyses of liquid biopsy that include testing for KRASG12C. Here, we performed a comprehensive analysis of KRASG12C mutations in multiple cancer types, as detected by circulating tumor DNA. METHODS: We conducted a 5-year retrospective review of KRASG12C mutations in patients with cancer who had undergone Guardant360 testing between July 1, 2014, and June 30, 2019; our study included treatment-naive and previously treated patients with metastatic solid tumors. RESULTS: KRASG12C mutations were identified in 2,985 of 80,911 patients (3.7%), across > 40 tumor types. KRASG12C mutations were detected most frequently in patients with nonsquamous non-small-cell lung cancer (NSCLC; 7.5%), NSCLC of all subtypes (6.9%), cancer of unknown primary (4.1%), colorectal cancer (3.5%), squamous NSCLC (2.0%), pulmonary neuroendocrine tumors (1.9%), and pancreatic ductal adenocarcinoma (1.2%) and cholangiocarcinoma (1.2%). KRASG12C mutations were predominantly clonal (clonality > 0.9%) in patients with lung adenocarcinoma, non-NSCLC, cancer of unknown primary, NSCLC, and pancreatic ductal adenocarcinoma, and patients with colorectal cancer and breast cancer had bimodal distribution of clonal and subclonal KRASG12C mutations. CONCLUSION: Our study demonstrates the feasibility of using circulating tumor DNA to identify KRASG12C mutations across solid tumors; the highest detection rate was in lung cancer, as previously reported in the literature.

Genomic profile of advanced breast cancer in circulating tumour DNA.

The genomics of advanced breast cancer (ABC) has been described through tumour tissue biopsy sequencing, although these approaches are limited by geographical and temporal heterogeneity. Here we use plasma circulating tumour DNA sequencing to interrogate the genomic profile of ABC in 800 patients in the plasmaMATCH trial. We demonstrate diverse subclonal resistance mutations, including enrichment of HER2 mutations in HER2 positive disease, co-occurring ESR1 and MAP kinase pathway mutations in HR + HER2- disease that associate with poor overall survival (p = 0.0092), and multiple PIK3CA mutations in HR + disease that associate with short progression free survival on fulvestrant (p = 0.0036). The fraction of cancer with a mutation, the clonal dominance of a mutation, varied between genes, and within hotspot mutations of ESR1 and PIK3CA. In ER-positive breast cancer subclonal mutations were enriched in an APOBEC mutational signature, with second hit PIK3CA mutations acquired subclonally and at sites characteristic of APOBEC mutagenesis. This study utilises circulating tumour DNA analysis in a large clinical trial to demonstrate the subclonal diversification of pre-treated advanced breast cancer, identifying distinct mutational processes in advanced ER-positive breast cancer, and novel therapeutic opportunities.

A Blood-based Assay for Assessment of Tumor Mutational Burden in First-line Metastatic NSCLC Treatment: Results from the MYSTIC Study.

PURPOSE: Tumor mutational burden (TMB) has been shown to be predictive of survival benefit in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors. Measuring TMB in the blood (bTMB) using circulating cell-free tumor DNA (ctDNA) offers practical advantages compared with TMB measurement in tissue (tTMB); however, there is a need for validated assays and identification of optimal cutoffs. We describe the analytic validation of a new bTMB algorithm and its clinical utility using data from the phase III MYSTIC trial. PATIENTS AND METHODS: The dataset used for the clinical validation was from MYSTIC, which evaluated first-line durvalumab (anti-PD-L1 antibody) ± tremelimumab (anticytotoxic T-lymphocyte-associated antigen-4 antibody) or chemotherapy for metastatic NSCLC. bTMB and tTMB were evaluated using the GuardantOMNI and FoundationOne CDx assays, respectively. A Cox proportional hazards model and minimal P value cross-validation approach were used to identify the optimal bTMB cutoff. RESULTS: In MYSTIC, somatic mutations could be detected in ctDNA extracted from plasma samples in a majority of patients, allowing subsequent calculation of bTMB. The success rate for obtaining valid TMB scores was higher for bTMB (809/1,001; 81%) than for tTMB (460/735; 63%). Minimal P value cross-validation analysis confirmed the selection of bTMB ≥20 mutations per megabase (mut/Mb) as the optimal cutoff for clinical benefit with durvalumab + tremelimumab. CONCLUSIONS: Our study demonstrates the feasibility, accuracy, and reproducibility of the GuardantOMNI ctDNA platform for quantifying bTMB from plasma samples. Using the new bTMB algorithm and an optimal bTMB cutoff of ≥20 mut/Mb, high bTMB was predictive of clinical benefit with durvalumab + tremelimumab versus chemotherapy.

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.

Spectrum of driver mutations and clinical impact of circulating tumor DNA analysis in non-small cell lung cancer: Analysis of over 8000 cases.

BACKGROUND: Circulating cell-free tumor DNA (ctDNA)-based mutation profiling, if sufficiently sensitive and comprehensive, can efficiently identify genomic targets in advanced lung adenocarcinoma. Therefore, the authors investigated the accuracy and clinical utility of a commercially available digital next-generation sequencing platform in a large series of patients with non-small cell lung cancer (NSCLC). METHODS: Plasma-based comprehensive genomic profiling results from 8388 consecutively tested patients with advanced NSCLC were analyzed. Driver and resistance mutations were examined with regard to their distribution, frequency, co-occurrence, and mutual exclusivity. RESULTS: Somatic alterations were detected in 86% of samples. The median variant allele fraction was 0.43% (range, 0.03%-97.62%). Activating alterations in actionable oncogenes were identified in 48% of patients, including EGFR (26.4%), MET (6.1%), and BRAF (2.8%) alterations and fusions (ALK, RET, and ROS1) in 2.3%. Treatment-induced resistance mutations were common in this cohort, including driver-dependent and driver-independent alterations. In the subset of patients who had progressive disease during EGFR therapy, 64% had known or putative resistance alterations detected in plasma. Subset analysis revealed that ctDNA increased the identification of driver mutations by 65% over standard-of-care, tissue-based testing at diagnosis. A pooled data analysis on this plasma-based assay demonstrated that targeted therapy response rates were equivalent to those reported from tissue analysis. CONCLUSIONS: Comprehensive ctDNA analysis detected the presence of therapeutically targetable driver and resistance mutations at the frequencies and distributions predicted for the study population. These findings add support for comprehensive ctDNA testing in patients who are incompletely tested at the time of diagnosis and as a primary option at the time of progression on targeted therapies.

Neurofibromin Is an Estrogen Receptor-α Transcriptional Co-repressor in Breast Cancer.

We report that neurofibromin, a tumor suppressor and Ras-GAP (GTPase-activating protein), is also an estrogen receptor-α (ER) transcriptional co-repressor through leucine/isoleucine-rich motifs that are functionally independent of GAP activity. GAP activity, in turn, does not affect ER binding. Consequently, neurofibromin depletion causes estradiol hypersensitivity and tamoxifen agonism, explaining the poor prognosis associated with neurofibromin loss in endocrine therapy-treated ER+ breast cancer. Neurofibromin-deficient ER+ breast cancer cells initially retain sensitivity to selective ER degraders (SERDs). However, Ras activation does play a role in acquired SERD resistance, which can be reversed upon MEK inhibitor addition, and SERD/MEK inhibitor combinations induce tumor regression. Thus, neurofibromin is a dual repressor for both Ras and ER signaling, and co-targeting may treat neurofibromin-deficient ER+ breast tumors.

Baseline Plasma Tumor Mutation Burden Predicts Response to Pembrolizumab-based Therapy in Patients with Metastatic Non-Small Cell Lung Cancer.

PURPOSE: The role of plasma-based tumor mutation burden (pTMB) in predicting response to pembrolizumab-based first-line standard-of-care therapy for metastatic non-small cell lung cancer (mNSCLC) has not been explored. EXPERIMENTAL DESIGN: A 500-gene next-generation sequencing panel was used to assess pTMB. Sixty-six patients with newly diagnosed mNSCLC starting first-line pembrolizumab-based therapy, either alone or in combination with chemotherapy, were enrolled (Clinicaltrial.gov identifier: NCT03047616). Response was assessed using RECIST 1.1. Associations were made for patient characteristics, 6-month durable clinical benefit (DCB), progression-free survival (PFS), and overall survival (OS). RESULTS: Of 66 patients, 52 (78.8%) were pTMB-evaluable. Median pTMB was 16.8 mutations per megabase (mut/Mb; range, 1.9-52.5) and was significantly higher for patients achieving DCB compared with no durable benefit (21.3 mut/Mb vs. 12.4 mut/Mb, P = 0.003). For patients with pTMB ≥ 16 mut/Mb, median PFS was 14.1 versus 4.7 months for patients with pTMB < 16 mut/Mb [HR, 0.30 (0.16-0.60); P < 0.001]. Median OS for patients with pTMB ≥ 16 was not reached versus 8.8 months for patients with pTMB < 16 mut/Mb [HR, 0.48 (0.22-1.03); P = 0.061]. Mutations in ERBB2 exon 20, STK11, KEAP1, or PTEN were more common in patients with no DCB. A combination of pTMB ≥ 16 and absence of negative predictor mutations was associated with PFS [HR, 0.24 (0.11-0.49); P < 0.001] and OS [HR, 0.31 (0.13-0.74); P = 0.009]. CONCLUSIONS: pTMB ≥ 16 mut/Mb is associated with improved PFS after first-line standard-of-care pembrolizumab-based therapy in mNSCLC. STK11/KEAP1/PTEN and ERBB2 mutations may help identify pTMB-high patients unlikely to respond. These results should be validated in larger prospective studies.

Facing the challenge of genetic counselors' need for rapid continuing education about genomic technologies.

The last couple of decades have seen the rapid advancement of genomic technologies (GT) and their equally rapid adoption into clinical testing. Regardless of specialty, all genetic counselors are unified by the fundamental goal to aid in diagnosing patient's genetic disease underscoring the importance for genetic counselors to maintain an in-depth understanding of GT. The National Society of Genetic Counselors' (NSGC) GT Special Interest Group conducted an online survey of NSGC members to assess current genomic technologies knowledge gaps. A total of 171 individuals from a variety of primary work settings completed the survey sufficiently to be included in the analysis. The majority of respondents received their degree in genetic counseling in more recent years (2000-2015). On average across all technologies, >70% of respondents deemed knowledge of GTs as important for successful job performance, 55% responded that additional job training in GTs is needed to successfully perform job functions, and only 28% responded that graduate training in GTs was good. Overall, the data show that participating genetic counselors perceive that their knowledge of GTs is inadequate while it is a key component of their jobs. These results have implications both for training programs and for continuing education efforts. These data can be used as a starting point for additional research into GT educational needs of genetic counselors.

Next generation sequencing of cell free circulating tumor DNA in blood samples of recurrent and metastatic head and neck cancer patients.

BACKGROUND: Effective targeted therapy is lacking in head and neck cancer (HNC). The use of next generation sequencing (NGS) has been suggested as a way to potentially expand therapeutic options and improve outcomes. This study was performed in order to further characterize blood sample cell-free circulating tumor DNA (ctDNA) in advanced HNC patients, to determine its ability to identify actionable mutations, and to elucidate its potential role in patient management. METHODS: Retrospective analysis of 60 patients with recurrent and metastatic (R/M) HNCs who underwent molecular profiling of blood samples utilizing Guardant360, a 70-gene ctDNA NGS platform. ctDNA sequencing data was compared to tumor NGS data, when available. Best response to therapy was assessed using RECIST measures. RESULTS: The most common tumor type was oropharyngeal squamous cell carcinoma (n=21). Other cancer types included salivary gland (n=8) and thyroid (n=4). The most common mutations identified by blood analysis were TP53 (68% of patients), PIK3CA (34% of patients), NOTCH1 (20% of patients), and ARID1A (15% of patients). These findings were consistent with results from tumor sequencing data (n=30) where TP53 (48%) and PIK3CA (24%) were also the most common. Seventy-three percent (n=22) of patients had alterations identified in blood that were not present in tumor specimens. In patients with squamous cell carcinoma, 66% had an off-label option identified and 90% had a trial option identified, while 50% of patients with salivary primaries had off-label option identified and 75% had trial options identified. All patients (n=3, 100%) with thyroid primaries had off-label and clinical trial options identified. Of patients with actionable mutations, 13% (n=8) received matched targeted therapy (MTT). Three patients had stable disease (37.5%), 3 had progressive disease (37.5%), and 2 (25%) were not evaluated at the time of follow up. Of those who did not receive targeted therapy (n=21), 11 patients had stable disease (52.4%), 9 had progressive disease (42.9%), and 1 had a complete response (4.8%). CONCLUSIONS: Alterations identified by ctDNA may help inform management decisions in advanced HNC. The majority of patients had unique mutations identified on ctDNA. The role of NGS of ctDNA should be explored in future studies.

Molecular Profiling of Hepatocellular Carcinoma Using Circulating Cell-Free DNA.

PURPOSE: Molecular profiling has been used to select patients for targeted therapy and determine prognosis. Noninvasive strategies are critical to hepatocellular carcinoma (HCC) given the challenge of obtaining liver tissue biopsies. EXPERIMENTAL DESIGN: We analyzed blood samples from 206 patients with HCC using comprehensive genomic testing (Guardant Health) of circulating tumor DNA (ctDNA). RESULTS: A total of 153/206 (74.3%) were men; median age, 62 years (range, 18-91 years). A total of 181/206 patients had ≥1 alteration. The total number of alterations was 680 (nonunique); median number of alterations/patient was three (range, 1-13); median mutant allele frequency (% cfDNA), 0.49% (range, 0.06%-55.03%). TP53 was the common altered gene [>120 alterations (non-unique)] followed by EGFR, MET, ARID1A, MYC, NF1, BRAF, and ERBB2 [20-38 alterations (nonunique)/gene]. Of the patients with alterations, 56.9% (103/181) had ≥1 actionable alterations, most commonly in MYC, EGFR, ERBB2, BRAF, CCNE1, MET, PIK3CA, ARID1A, CDK6, and KRAS. In these genes, amplifications occurred more frequently than mutations. Hepatitis B (HBV)-positive patients were more likely to have ERBB2 alterations, 35.7% (5/14) versus 8.8% HBV-negative (P = 0.04). CONCLUSIONS: This study represents the first large-scale analysis of blood-derived ctDNA in HCC in United States. The genomic distinction based on HCC risk factors and the high percentage of potentially actionable genomic alterations suggests potential clinical utility for this technology.

Validation of Microsatellite Instability Detection Using a Comprehensive Plasma-Based Genotyping Panel.

PURPOSE: To analytically and clinically validate microsatellite instability (MSI) detection using cell-free DNA (cfDNA) sequencing. EXPERIMENTAL DESIGN: Pan-cancer MSI detection using Guardant360 was analytically validated according to established guidelines and clinically validated using 1,145 cfDNA samples for which tissue MSI status based on standard-of-care tissue testing was available. The landscape of cfDNA-based MSI across solid tumor types was investigated in a cohort of 28,459 clinical plasma samples. Clinical outcomes for 16 patients with cfDNA MSI-H gastric cancer treated with immunotherapy were evaluated. RESULTS: cfDNA MSI evaluation was shown to have high specificity, precision, and sensitivity, with a limit of detection of 0.1% tumor content. In evaluable patients, cfDNA testing accurately detected 87% (71/82) of tissue MSI-H and 99.5% of tissue microsatellite stable (863/867) for an overall accuracy of 98.4% (934/949) and a positive predictive value of 95% (71/75). Concordance of cfDNA MSI with tissue PCR and next-generation sequencing was significantly higher than IHC. Prevalence of cfDNA MSI for major cancer types was consistent with those reported for tissue. Finally, robust clinical activity of immunotherapy treatment was seen in patients with advanced gastric cancer positive for MSI by cfDNA, with 63% (10/16) of patients achieving complete or partial remission with sustained clinical benefit. CONCLUSIONS: cfDNA-based MSI detection using Guardant360 is highly concordant with tissue-based testing, enabling highly accurate detection of MSI status concurrent with comprehensive genomic profiling and expanding access to immunotherapy for patients with advanced cancer for whom current testing practices are inadequate.See related commentary by Wang and Ajani, p. 6887.

Detection of ERBB2 (HER2) Gene Amplification Events in Cell-Free DNA and Response to Anti-HER2 Agents in a Large Asian Cancer Patient Cohort.

Background: HER2 antagonists have marked activity and are approved for the treatment of HER2 overexpressing breast and gastric cancers. Recent studies have shown that ERBB2 (HER2) gene amplification and overexpression may also be actionable in other tumor types. Inter- and intratumoral heterogeneity in HER2 status, however, poses a significant challenge in identifying patients that may benefit from HER2-targeted therapies. ERBB2 amplification as identified by circulating cell-free DNA (cfDNA), which circumvents tissue heterogeneity issues, is emerging as a robust biomarker predictive of response to anti-HER2 agents. Here, the prevalence and genomic landscape of ERBB2 alterations detectable by next-generation sequencing (NGS) of cfDNA was evaluated in a large cohort of Asian patients with advanced solid tumors. Methods: Results were queried for consecutive patients (n = 469) tested by a comprehensive 70/73-gene cfDNA NGS assay (Guardant360®) between November 2015 and June 2018. Patients with ERBB2 gene alterations including copy number amplifications (CNAs), single nucleotide variants (SNVs), and insertion-deletions (indels) were identified. Results: ERBB2 alterations were detected in 52 patients (11.1%); ERBB2 SNVs, CNAs, and indels were found in 27 (5.8%), 27 (5.8%), and 10 (2.1%) patients, respectively. ERBB2 amplification was most frequently identified in gastric (21.4%; 6/28), colorectal (11.1%; 5/45), lung (3.9%; 9/231), and breast (3.2%; 1/31) cancer patients. ERBB2 amplification was often mutually exclusive with other oncogenic alterations in gastric (83.3%; 5/6) and colorectal (60%; 3/5) cancer patients. ERBB2 copy number gains were also highest in gastric and colorectal cancers (median 4.8 and 6.6, respectively). We further report two cases of advanced gastric cancer patients, one treatment naïve, and the other having failed four lines of therapy, whose ERBB2 CNAs were identified by cfDNA and derived clinical benefit from HER2-based therapies. Conclusion: Our data indicate that ERBB2 amplification is a common event in solid tumors among Asian cancer patients. High ERBB2 incidence and copy number gains were observed in gastric and colorectal cancer patients, often in the absence of other oncogenic mutations, underscoring its likely role as the driver alteration in those settings. Finally, we show the potential of comprehensive cfDNA testing in identifying patients who are most likely to benefit from HER2-targeted therapies.

Analysis of cell-free circulating tumor DNA in 419 patients with glioblastoma and other primary brain tumors.

Aim: Genomically matched trials in primary brain tumors (PBTs) require recent tumor sequencing. We evaluated whether circulating tumor DNA (ctDNA) could facilitate genomic interrogation in these patients. Methods: Data from 419 PBT patients tested clinically with a ctDNA NGS panel at a CLIA-certified laboratory were analyzed. Results: A total of 211 patients (50%) had ≥1 somatic alteration detected. Detection was highest in meningioma (59%) and gliobastoma (55%). Single nucleotide variants were detected in 61 genes, with amplifications detected in ERBB2, MET, EGFR and others. Conclusion: Contrary to previous studies with very low yields, we found half of PBT patients had detectable ctDNA with genomically targetable off-label or clinical trial options for almost 50%. For those PBT patients with detectable ctDNA, plasma cfDNA genomic analysis is a clinically viable option for identifying genomically driven therapy options.

Targeted Tissue and Cell-Free Tumor DNA Sequencing of Advanced Lung Squamous-Cell Carcinoma Reveals Clinically Significant Prevalence of Actionable Alterations.

BACKGROUND: Major guidelines do not recommend routine molecular profiling of lung squamous-cell carcinoma (LUSC) because the prevalence of actionable alterations is thought to be low. Increased utilization of next-generation sequencing (NGS), particularly with cell-free circulating tumor DNA, facilitates reevaluation of this premise. PATIENTS AND METHODS: We retrospectively evaluated the prevalence of actionable alterations in 2 distinct LUSC cohorts totaling 492 patients. A total of 410 consecutive patients with stage 3B or 4 LUSC were tested with a targeted cell-free circulating DNA NGS assay, and 82 patients with LUSC of any stage were tested with a tissue NGS cancer panel. RESULTS: In the overall cohort, 467 patients (94.9%) had a diagnosis of LUSC, and 25 patients (5.1%) had mixed histology with a squamous component. A total of 10.5% of the LUSC subgroup had somatic alterations with therapeutic relevance, including in EGFR (2.8%), ALK/ROS1 (1.3%), BRAF (1.5%), and MET amplification or exon 14 skipping (5.1%). Sixteen percent of patients with mixed histology had an actionable alteration. In the LUSC subgroup, 3 evaluable patients were treated with targeted therapy for an actionable alteration; all of them experienced partial response. CONCLUSION: In this large, real-world LUSC cohort, we observed a clinically significant prevalence of actionable alterations. Accurate local histopathologic assessment in advanced-stage LUSC can be challenging. Further evaluation of the genomic landscape in this setting is warranted to potentially identify underappreciated treatment options.

Identification of Incidental Germline Mutations in Patients With Advanced Solid Tumors Who Underwent Cell-Free Circulating Tumor DNA Sequencing.

PURPOSE: To determine the potential for detection of incidental germline cancer predisposition mutations through cell-free DNA (cfDNA) analyses in patients who underwent solid tumor somatic mutation evaluation. PATIENTS AND METHODS: Data were evaluated from 10,888 unselected patients with advanced (stage III/IV) cancer who underwent Guardant360 testing between November 2015 and December 2016. The main outcome was prevalence of putative germline mutations identified among 16 actionable hereditary cancer predisposition genes. RESULTS: More than 50 cancer types were studied, including lung (41%), breast (19%), colorectal (8%), prostate (6%), pancreatic (3%), and ovarian (2%). Average patient age was 63.5 years (range, 18 to 95 years); 43% were male. One hundred and fifty-six individuals (1.4%) had suspected hereditary cancer mutations in 11 genes. Putative germline mutations were more frequent in individuals younger than 50 years versus those 50 years and older (3.0% v 1.2%, respectively; P < .001). Highest yields of putative germline findings were in patients with ovarian (8.13%), prostate (3.46%), pancreatic (3.34%), and breast (2.2%) cancer. Putative germline mutation identification was consistent among 12 individuals with multiple samples. Patients with circulating tumor DNA copy number variation and/or reversion mutations suggestive of functional loss of the wild-type allele in the tumor DNA also are described. CONCLUSION: Detection of putative germline mutations from cfDNA is feasible across multiple genes and cancer types without prior mutation knowledge. Many mutations were found in cancers without clear guidelines for hereditary cancer genetic counseling/testing. Given the clinical significance of identifying hereditary cancer predisposition for patients and their families as well as targetable germline alterations such as in BRCA1 or BRCA2, research on the best way to validate and return potential germline results from cfDNA analysis to clinicians and patients is needed.

Molecular Landscape of ERBB2/ERBB3 Mutated Colorectal Cancer.

Background: Despite growing therapeutic relevance of ERBB2 amplifications in colorectal cancer (CRC), little is known about ERBB2/ERBB3 mutations. We aimed to characterize these subsets of CRC. Methods: We performed a retrospective analysis of 419 CRC patients from MD Anderson (MDACC) and 619 patients from the Nurses' Health Study (NHS)/Health Professionals Follow-Up Study (HPFS) with tissue sequencing, clinicopathologic, mutational, and consensus molecular subtype (CMS) profiles of ERBB2/ERBB3 mutant patients. A third cohort of 1623 CRC patients with ctDNA assays characterized the ctDNA profile of ERBB2 mutants. All statistical tests were two-sided. Results: ERBB2 mutations occurred in 4.1% (95% confidence interval [CI] = 2.4% to 6.4%), 5.8% (95% CI = 4.1% to 8.0%), and 5.1% (95% CI = 4.0% to 6.2%) of MDACC, NHS/HPFS, and ctDNA patients, respectively. ERBB3 mutations occurred in 5.7% (95% CI = 3.7% to 8.4%, 95% CI = 4.0% to 7.8%) of patients in both tissue cohorts. Age, stage, and tumor location were not associated with either mutation. Microsatellite instability (MSI) was associated with ERBB2 (odds ratio [OR] = 5.98, 95% CI = 2.47 to 14.49, P < .001; OR = 5.13, 95% CI = 2.38 to 11.05, P < .001) and ERBB3 mutations (OR = 3.48, 95% CI = 1.51 to 8.02, P = .002; OR = 3.40, 95% CI = 1.05 to 10.96, P = .03) in both tissue cohorts. Neither gene was associated with TP53, APC, KRAS, NRAS, or BRAF mutations in tissue. However, PIK3CA mutations were strongly associated with ERBB2 mutations in all three cohorts (OR = 3.68, 95% CI = 1.83 to 7.41, P = .001; OR = 2.25, 95% CI = 1.11 to 4.58, P = .02; OR = 2.11, 95% CI = 1.25 to 3.58, P = .004) and ERBB3 mutations in the MDACC cohort (OR = 13.26, 95% CI = 5.27 to 33.33, P < .001). ERBB2 (P = 0.08) and ERBB3 (P = .008) mutations were associated with CMS1 subtype. ERBB2 (hazard ratio [HR] = 1.82, 95% CI = 1.23 to 4.03, P = .009), but not ERBB3 (HR = 0.88, 95% CI = 0.45 to 1.73, P = .73), mutations were associated with worse overall survival. Conclusions: MSI and PIK3CA mutations are associated with ERBB2/ERBB3 mutations. Co-occurring PIK3CA mutations may represent a second hit to oncogenic signaling that needs consideration when targeting ERBB2/ERBB3.

The Landscape of Actionable Genomic Alterations in Cell-Free Circulating Tumor DNA from 21,807 Advanced Cancer Patients.

Purpose: Cell-free DNA (cfDNA) sequencing provides a noninvasive method for obtaining actionable genomic information to guide personalized cancer treatment, but the presence of multiple alterations in circulation related to treatment and tumor heterogeneity complicate the interpretation of the observed variants.Experimental Design: We describe the somatic mutation landscape of 70 cancer genes from cfDNA deep-sequencing analysis of 21,807 patients with treated, late-stage cancers across >50 cancer types. To facilitate interpretation of the genomic complexity of circulating tumor DNA in advanced, treated cancer patients, we developed methods to identify cfDNA copy-number driver alterations and cfDNA clonality.Results: Patterns and prevalence of cfDNA alterations in major driver genes for non-small cell lung, breast, and colorectal cancer largely recapitulated those from tumor tissue sequencing compendia (The Cancer Genome Atlas and COSMIC; r = 0.90-0.99), with the principal differences in alteration prevalence being due to patient treatment. This highly sensitive cfDNA sequencing assay revealed numerous subclonal tumor-derived alterations, expected as a result of clonal evolution, but leading to an apparent departure from mutual exclusivity in treatment-naïve tumors. Upon applying novel cfDNA clonality and copy-number driver identification methods, robust mutual exclusivity was observed among predicted truncal driver cfDNA alterations (FDR = 5 × 10-7 for EGFR and ERBB2), in effect distinguishing tumor-initiating alterations from secondary alterations. Treatment-associated resistance, including both novel alterations and parallel evolution, was common in the cfDNA cohort and was enriched in patients with targetable driver alterations (>18.6% patients).Conclusions: Together, these retrospective analyses of a large cfDNA sequencing data set reveal subclonal structures and emerging resistance in advanced solid tumors. Clin Cancer Res; 24(15); 3528-38. ©2018 AACR.

Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies.

Purpose: To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility.Experimental Design: Analytic validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples.Results: Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24-2.76 copies 95% limits of detection while maintaining high specificity [prevalence-adjusted positive predictive values (PPV) >98%]. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity [positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%-100%]. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non-small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker.Conclusions: High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery. Clin Cancer Res; 24(15); 3539-49. ©2018 AACR.