Lanman, Richard B.

AbstractPurpose: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

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer death among men and women in the United States, and additional treatment strategies are needed for patients with advanced disease. Prospective therapeutic studies based on tissue-detected genomic targets such as ERBB2 (HER2), BRAF V600E, gene rearrangements/fusions, and microsatellite instability (MSI) have demonstrated clinical benefit for mCRC patients. Although correlative studies and/or retrospective case series utilizing circulating cell-free DNA (cfDNA) next generation sequencing (NGS) have shown that plasma detection of these genomic targets shows promise in mCRC, prospective studies are lacking. We hypothesize that comprehensive genomic profiling of cfDNA will identify actionable targets and improve outcomes for patients with mCRC. Methods: COLOMATE (Colorectal Cancer and Liquid Biopsy Screening Protocol for Molecularly Assigned Therapy; NCT03765736) is a phase II umbrella screening protocol sponsored by the Academic and Community Cancer Research United (ACCRU) consortium. This study utilizes Guardant360™ (Guardant Health, Redwood City, CA) - a blood-based targeted NGS panel - to identify actionable genomic alterations in mCRC patients and to assess the impact of molecularly assigned therapy accordingly under its individual companion studies. Eligible mCRC patients must have progressed on, been intolerant to, or have a contraindication to treatment with a fluoropyrimidine, oxaliplatin, irinotecan, anti-vascular endothelial growth factor (VEGF) monoclonal antibodies (mAbs) and anti-epidermal growth factor receptor (EGFR) mAbs if RAS wild-type. Companion studies are dynamic and currently include those for patients whose tumors are either RAS/BRAF wild-type, HER2 amplified, FGFR altered, RAS mutated, or without any known actionable alteration. Additional companion studies are under active development. The primary objectives of this study are to perform blood-based genomic profiling on patients with treatment refractory mCRC to facilitate accrual to molecularly assigned therapies, and to obtain patient-matched tumor tissue and cell free DNA from peripheral blood to facilitate clinically annotated genomic analyses. Secondary correlative objectives are to explore mechanisms of acquired resistance to molecularly assigned therapy and to explore the correlation between cfDNA mutational burden (allele frequency, copy number) and clinical outcomes such as objective response rate, progression-free survival, and overall survival. Citation Format: Kristen K. Ciombor, Fang-Shu Ou, Andrew Dodge, Tyler Zemla, Christina Wu, Kimmie Ng, Katrina Pedersen, Shumei Kato, Pashtoon M. Kasi, Daniel Ahn, Rebecca Nagy, Richard Lanman, Scott Kopetz, John H. Strickler, Tanios Bekaii-Saab. COLOMATE: Colorectal cancer and liquid biopsy screening protocol for molecularly assigned therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-235.

4070 Background: Esophageal cancer (EC) is a lethal malignancy with limited treatment options. Genomic analyses have led to the elucidation of numerous dysregulated genes in esophageal adenocarcinoma (AC) and squamous cell carcinoma (SCC), and the potential for advancement of targeted therapies in this disease. Data regarding circulating tumor DNA (ctDNA) plasma analysis in EC in real-world clinical practice is limited. Methods: We performed ctDNA next-generation sequencing (NGS) analysis in patients (pts) with EC (January 2015- February 2018). ctDNA analysis was performed using Guardant 360 (Guardant Health, CA) which detects single nucleotide variants and insertion/deletion mutations, and specific amplifications and fusions, in up to 73 different genes. The mutant allele fraction (MAF) for detected alterations was calculated relative to wild type in ctDNA. Therapeutic relevance was defined as alterations within OncoKB levels 1-3B and R1. Results: Among 450 pts, 487 total samples were analyzed (77% AC, 31% SCC). ctDNA NGS revealed at least one genomic alteration (excluding variants of uncertain significance and synonymous mutations) in 81% of pts (90% AC, 88% SCC). Median number of alterations per AC patient was 4 [range, 1-59] and a median MAF of 0.84% (range, 0.02% - 83.7%); SCC was 5 [range, 1-26], with a median MAF of 0.99% (range, 0.01% - 85.2%). The total number of unique alterations was 1,162. The most commonly altered genes in AC: TP53 (70%), KRAS (20%), ERBB2 (18%), EGFR (16%), PIK3CA (16%); in SCC: TP53 (88%), PIK3CA (24%), CCND1 (23%), KRAS (21%), EGFR 15%). Therapeutically relevant alterations will be described. Conclusions: ctDNA plasma profiling of pts with EC is a feasible alternative and non-invasive method to gather comprehensive genomic data. Further large comparison studies to assess landscape of genomic alterations observed through ctDNA versus tissue-based assays, in addition to studies of targeted therapy outcomes based on ctDNA-detected alterations, are needed.

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.

Abstract Background: Targeted therapies are increasingly under evaluation in clinical trials for the treatment of biliary tract cancer (BTC), and cell-free tumor DNA assays are an emerging technology for the detection of actionable alterations. Methods: ctDNA analysis was performed in patients with BTC using the CLIA-certified Guardant360 liquid biopsy assay as part of routine clinical care. The assay screens up to 73 genes for single-nucleotide variants (SNVs), 23 for insertions or deletions (INDELs), 18 for copy number variations (CNVs), and 6 for fusions. Results: 855 samples were analyzed from 751 unique BTC patients. Median age was 64 years (range, 29-89), 53% were female, and 631 (84%) had at least one alteration in their first sample. In the 751 patients, the most common genes altered were TP53 (39%), KRAS (15%), PIK3CA (13%), ARID1A (13%), EGFR (11%), FGFR2 (11%), ERBB2 (11%), NF1 (10%), IDH1 (10%), APC (9%), BRAF (9%), MYC (8%), MET (7%), CCNE1 (7%), and FGFR1 (7%). Alterations in DNA damage response genes were also seen, including BRCA2 (6%), BRCA1 (5%), ATM (4%), and MLH1 (1%). The median number of alterations per sample was 3 (range, 0-40). In a clinically annotated dataset from the Massachusetts General Hospital Cancer Center, 112 samples were obtained from 61 unique patients. Among these, 52 (85%) had intrahepatic cholangiocarcinoma (ICC), 3 (5%) had extrahepatic cholangiocarcinoma, and 6 (10%) had gallbladder cancer. 20/61 (33%) patients were treated with targeted therapy. Among 20 patients with both ctDNA and tumor sequencing results, the targetable mutation was found in both ctDNA and tumor tissue in 7/20 (35%), solely in ctDNA in 1/20 (5%), and solely in tissue in 12/20 (60%) patients. The patient with the alteration detected solely in ctDNA had insufficient tissue for analysis; she was found to have an FGFR2 F276C mutation and benefited for 8.5 months from an FGFR inhibitor on a clinical trial. Of the 12 patients whose targetable mutation was not detected in plasma, 10 (83%) had an FGFR2 fusion, 1 had an INDEL in FGFR2 (Leu262_Ala266delinsAsp), and 1 had an FGFR3 splice site mutation. 15/20 (75%) patients treated with targeted therapy were followed with serial ctDNA analysis, and a potential resistance mechanism was identified in 5/15 (33%). All 5 patients had FGFR2-fusion positive ICC and developed mutations in the FGFR2 kinase domain as a potential resistance mechanism to FGFR inhibition, including the FGFR2 V564F gatekeeper mutation in 4/5 patients. Conclusions: In patients with BTC, ctDNA-based genomic profiling can detect alterations in IDH, FGFR, PIK3CA, ERBB2, BRAF, and MET. The technology is still evolving for the detection of FGFR2 fusions, which is hindered in capture-based methods in plasma by the large number of unique breakpoints and fusion partners. ctDNA analysis may also provide a noninvasive mechanism to study resistance to targeted therapy and clonal dynamics in BTC. Citation Format: Lipika Goyal, Robin Kate Kelley, Lesli Kiedrowski, Daniel Catenacci, Kabir Mody, Rachna Shroff, Aparna Parikh, Stephanie Reyes, Jordan R. Maurer, Ryan B. Corcoran, Paul Fanta, Mike Cusnir, Becky Nagy, Richard Lanman, Michael Morse, Benjamin Tan, Milind Javle, Andrew Zhu. Blood-based genomic profiling of cell-free tumor DNA (ctDNA) in patients with biliary tract cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A183.

Abstract Introduction: MutHER is a phase II trial that demonstrated the anti-tumor activity of the pan-HER inhibitor neratinib in HER2mut, non-amplified MBC. The major challenges to accrue to this trial were the large number of pts to screen for the 2-3% HER2mut population and the high rate (24%) of poor quality tumor DNA for sequencing. The goals of this ctDNA study were: 1) the concordance of HER2mut detected by ctDNA versus tumor testing; 2) the incidence of ctDNA HER2mut in HER2 non-amplified MBC; 3) changes in HER2mut variant allele frequency (VAF) on neratinib therapy. Methods: A sample size of 30 negative (neg) controls was needed to ensure 90% confidence if ctDNA testing has >90% specificity in detecting HER2mut. Thus, plasma from MBC pts obtained at screening for MutHER trial (Neg control: 40 pts without HER2mut on tumor testing; Positive (pos) control: 14 pts with known HER2mut who received neratinib) were subjected to Guardant360 ctDNA 70-gene panel sequencing (all exons of HER2 included). ctDNA from the 14 neratinib treated pts were also analyzed at week (wk) 4 and upon progression. ctDNA data from MBC pts clinically tested at Guardant Health were interrogated for HER2mut incidence. Results: Among the 14 pts with tumor pos for HER2mut, ctDNA sequencing identified the same HER2mut in 11, discrepant HER2mut in 1, and neg in 2. The 2 pts with ctDNA neg for HER2mut had progressive disease (PD) and stable disease (SD > 6 months) on neratinib, respectively. Among the 40 neg controls, 8 were not evaluable (no detectable ctDNA or assay unsuccessful) and all 32 successfully sequenced cases were neg for HER2mut. The sensitivity and specificity of ctDNA for HER2mut detection was 11/14 (79%, 90% CI: 53-94%) and 32/32 (100%, 90% CI: 91-100%), respectively. Among the 11 paired baseline and wk 4 samples, 9 (82%) had lower HER2mut VAFs at wk 4 than at baseline, with 1 complete response (CR), 1 partial response (PR), 5 SD, and 4 PD at wk 8 as best tumor response. Two pts had higher wk 4 ctDNA HER2mut VAFs and both had radiographic PD at wk 8. The absolute HER2mut VAF levels at wk 4 were significantly associated with TTP (Spearman rho=-0.69, p=0.02) and tumor size change (rho=0.67, p=0.05). The HER2mut VAFs were significantly higher at progression than wk 4 in all pts (p<0.01). One pt acquired a new HER2mut T798I, which is analogous to the gate-keeper mutation EGFR T790M. The incidence of HER2mut without amplification in unselected consecutive MBC clinically tested by Guardant360 was 3% (48/1,584), with mutation pattern similar to published tumor testing data. Conclusions: ctDNA sequencing is sensitive and highly specific in detecting HER2mut, offering a non-invasive method to identify pts for trials of HER2mut-targeted therapy. Decreased HER2mut VAFs at wk 4 was observed in 82% of cases, consistent with the on-target effect of neratinib. Increased HER2mut VAFs at wk 4 is a potential early marker of progression. Citation Format: Cynthia Ma, Ron Bose, Feng Gao, Rachel Freedman, Melinda Telli, Gretchen Kimmick, Eric Winer, Michael Naughton, Matthew Goetz, Christy Russell, Debu Tripathy, Melody Cobleigh, Andres Forero, Timothy Pluard, Carey Anders, Shana Thomas, Jill Anderson, Caroline Bumb, Kimberly Banks, Richard Lanman, Richard Bryce, Alshad Lalani, John Pfeifer, Daniel Hays, Mark Pegram, Kimberly Blackwell, Philippe Bedard, Hussam Al-Kateb, Matthew Ellis. Circulating tumor DNA (ctDNA) sequencing for HER2 mutation (HER2mut) screening and response monitoring to neratinib in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT011. doi:10.1158/1538-7445.AM2017-CT011

Abstract Purpose: Based on promising preclinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2mut detection. Experimental Design: Tumor tissue positive for HER2mut was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2mut). Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2mut cases were identified locally. Twenty-one of these 22 HER2mut cases were estrogen receptor positive. Sixteen patients [median age 58 (31–74) years and three (2–10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI), 13%–55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8–31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2mut in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%–94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%–100%). In addition, ctDNA HER2mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression. Conclusions: Neratinib is active in HER2mut, nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2mut cancers for clinical trial participation. Clin Cancer Res; 23(19); 5687–95. ©2017 AACR.

Background: Seventy-five percent of thyroid nodules with indeterminate fine-needle aspiration (FNA) cytology are found to be benign postoperatively. A novel genomic test, the Afirma ® gene expression classifier (AGEC), has been available for clinical use in the United States, since late 2010. In 2010, two modest-sized validation studies showed that the AGEC could identify a benign gene expression signature in indeterminate cytology thyroid FNA samples with a negative predictive value >95%. The objective of this study was to evaluate how the AGEC impacted the joint decision of the endocrinologist and patient to operate when FNA cytology was indeterminate, but the AGEC reading of the nodule was benign. Methods: In this cross-sectional cohort study, data were contributed retrospectively by 51 endocrinologists at 21 practice sites that had previously obtained ≥3 benign AGEC readings in ≥1 cm nodules with indeterminate FNA cytology readings. Information regarding demographic data, nodule size and location, decision to operate, surgery type (hemithyroidectomy [HT] or total thyroidectomy [TT]), and reason for recommending surgery was retrospectively collected. Results: Compared to a 74% previous historical rate of surgery for cytologically indeterminate nodules, the operative rate fell to 7.6% during the period that AGEC were obtained in the clinical practices, a highly significant reduction in the decision to operate ( p <0.001). The rate of surgery on cytologically indeterminate nodules that were benign by the AGEC reading did not differ from the historically reported rate of operation on cytologically benign nodules ( p =0.41). The four primary reasons reported by the physicians for operating on nodules with a benign AGEC reading, in descending order: large nodule size (46.4%), symptomatic nodules (25.0%), rapidly growing nodules (10.7%), or a second suspicious or malignant nodule in the same patient (10.7%). These reasons are concordant with those typically given for operation on cytologically benign nodules. Conclusions: In a substantial group of medical practices, obtaining an AGEC test in patients with cytologically indeterminate nodules was associated with a striking reduction in the rate of diagnostic thyroidectomy. Approximately, one surgery was avoided for every two AGEC tests run on thyroid FNAs with indeterminate cytology.