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OBJECTIVES: Effective therapy for non-small-cell lung cancer (NSCLC) depends on morphological and genomic classification, with comprehensive screening for guideline-recommended biomarkers critical to guide treatment. Companion diagnostics, which provide robust genotyping results, represent an important component of personalized oncology. We evaluated the clinical validity of Guardant360 CDx as a companion diagnostic for sotorasib for detection of KRAS p.G12C, an important oncogenic NSCLC driver mutation. MATERIALS AND METHODS: KRAS p.G12C was tested in NSCLC patients from CodeBreaK100 (NCT03600833) in pretreatment plasma samples using Guardant360 CDx liquid biopsy and archival tissue samples using therascreen® KRAS RGQ polymerase chain reaction (PCR) kit tissue testing. Matched tissue and plasma samples were procured from other clinical trials or commercial vendors, and results were compared. Demographics and clinical characteristics and objective response rate (ORR) were evaluated. RESULTS: Of 126 CodeBreaK patients, 112 (88.9%) were tested for KRASp.G12C mutations with Guardant360 CDx. Among 189 patients in the extended analysis cohort, the positive and negative percent agreement (95% CI) for Guardant360 CDx plasma testing relative to therascreen® KRAS RGQ PCR kit tissue testing were 0.71 (0.62, 0.79) and 1.00 (0.95, 1.00), respectively; overall percent agreement (95% CI) was 0.82 (0.76, 0.87). TP53 co-mutations were the most common regardless of KRAS p.G12C status (KRAS p.G12C-positive, 53.4%; KRAS p.G12C-negative, 45.5%). STK11 was co-mutated in 26.1% of KRAS p.G12C-positive samples. The ORR was similar among patients selected by plasma and tissue testing. CONCLUSION: Comprehensive genotyping for all therapeutic targets including KRAS p.G12C is critical for management of NSCLC. Liquid biopsy using Guardant360 CDx has clinical validity for identification of patients with KRASp.G12C-mutant NSCLC and, augmented by tissue testing methodologies as outlined on the approved product label, will identify patients for treatment with sotorasib. ispartof: Lung Cancer vol:166 pages:270-278 ispartof: location:Ireland status: published

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

The applicability of circulating tumor DNA (ctDNA) genotyping to inform enrollment of patients with cancer in clinical trials has not been established. We conducted a phase 2 trial to evaluate the efficacy of pertuzumab plus trastuzumab for metastatic colorectal cancer (mCRC), with human epidermal growth factor receptor 2 (HER2) amplification prospectively confirmed by tumor tissue or ctDNA analysis (UMIN000027887). HER2 amplification was confirmed in tissue and/or ctDNA in 30 patients with mCRC. The study met the primary endpoint with a confirmed objective response rate of 30% in 27 tissue-positive patients and 28% in 25 ctDNA-positive patients, as compared to an objective response rate of 0% in a matched real-world reference population treated with standard-of-care salvage therapy. Post hoc exploratory analyses revealed that baseline ctDNA genotyping of HER2 copy number and concurrent oncogenic alterations adjusted for tumor fraction stratified patients according to efficacy with similar accuracy to tissue genotyping. Decreased ctDNA fraction 3 weeks after treatment initiation associated with therapeutic response. Pertuzumab plus trastuzumab showed similar efficacy in patients with mCRC with HER2 amplification in tissue or ctDNA, showing that ctDNA genotyping can identify patients who benefit from dual-HER2 blockade as well as monitor treatment response. These findings warrant further use of ctDNA genotyping in clinical trials for HER2-amplified mCRC, which might especially benefit patients in first-line treatment., The multicenter phase 2 TRIUMPH trial shows the utility of ctDNA genotyping as a screening platform to select patients with HER2-amplified metastatic colorectal cancer who benefit from dual-HER2 blockade with trastuzumab and pertuzumab

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.

With advances in precision oncology, liquid biopsies have shown promise as a minimally invasive means to diagnose cancer and guide treatment decisions. This commentary presents an assessment of the potential and the challenges of widespread use of liquid biopsy testing, based on the 2019 Accelerating Anticancer Agent Development Workshop.

Purpose:Complete and timely tissue genotyping is challenging, leading to significant numbers of patients with newly diagnosed metastatic non–small cell lung cancer (mNSCLC) being undergenotyped for all eight genomic biomarkers recommended by professional guidelines. We aimed to demonstrate noninferiority of comprehensive cell-free DNA (cfDNA) relative to physician discretion standard-of-care (SOC) tissue genotyping to identify guideline-recommended biomarkers in patients with mNSCLC.Patients and Methods:Prospectively enrolled patients with previously untreated mNSCLC undergoing physician discretion SOC tissue genotyping submitted a pretreatment blood sample for comprehensive cfDNA analysis (Guardant360).Results:Among 282 patients, physician discretion SOC tissue genotyping identified a guideline-recommended biomarker in 60 patients versus 77 cfDNA identified patients (21.3% vs. 27.3%; P < 0.0001 for noninferiority). In tissue-positive patients, the biomarker was identified alone (12/60) or concordant with cfDNA (48/60), an 80% cfDNA clinical sensitivity for any guideline-recommended biomarker. For FDA-approved targets (EGFR, ALK, ROS1, BRAF) concordance was >98.2% with 100% positive predictive value for cfDNA versus tissue (34/34 EGFR-, ALK-, or BRAF-positive patients). Utilizing cfDNA, in addition to tissue, increased detection by 48%, from 60 to 89 patients, including those with negative, not assessed, or insufficient tissue results. cfDNA median turnaround time was significantly faster than tissue (9 vs. 15 days; P < 0.0001). Guideline-complete genotyping was significantly more likely (268 vs. 51; P < 0.0001).Conclusions:In the largest cfDNA study in previously untreated mNSCLC, a validated comprehensive cfDNA test identifies guideline-recommended biomarkers at a rate at least as high as SOC tissue genotyping, with high tissue concordance, more rapidly and completely than tissue-based genotyping.See related commentary by Meador and Oxnard, p. 4583

PARP1/2 inhibitors are effective against BRCA2-deficient tumors. The PARP inhibitor (PARPi) olaparib received FDA breakthrough designation for treatment of metastatic castration-resistant prostate cancers (CRPC) carrying mutations in BRCA1/2 or ATM genes. Emergent resistance to PARPi has been associated with tumor-specific BRCA2 mutations that revert the normal open reading frame rescuing homologous recombination. We describe a case of metastatic CRPC with germline BRCA2 mutation with acquired resistance to olaparib related to biallelic BRCA2 reversion mutations of both the germline and somatic loss of function alleles detected by circulating tumor DNA testing. We also summarize a retrospective analysis of 1,534 prostate cancer cases with ctDNA analysis showing a 1.6% incidence of germline BRCA2 mutations. Within the germline BRCA2-positive cases exposed to platinum chemotherapy or PARP inhibition, the prevalence of reversion mutations was 40%. This report documents the frequency of reversion mutations in a large cohort of prostate cancer patients carrying of BRCA mutations. It also shows the potential utility of ctDNA analyses for early detection of reversion mutation driving tumor resistance.

Background Schistosoma haematobium, the helminth causing urogenital schistosomiasis, is a known bladder carcinogen. Despite the causal link between S. haematobium and bladder cancer, the underlying mechanisms are poorly understood. S. haematobium oviposition in the bladder is associated with angiogenesis and urothelial hyperplasia. These changes may be pre-carcinogenic events in the bladder. We hypothesized that the Interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE), an S. haematobium egg-secreted “infiltrin” protein that enters host cell nuclei to alter cellular activity, is sufficient to induce angiogenesis and urothelial hyperplasia. Methods: Mouse bladders injected with S. haematobium eggs were analyzed via microscopy for angiogenesis and urothelial hyperplasia. Endothelial and urothelial cell lines were incubated with recombinant IPSE protein or an IPSE mutant protein that lacks the native nuclear localization sequence (NLS-) and proliferation measured using CFSE staining and real-time monitoring of cell growth. IPSE’s effects on urothelial cell cycle status was assayed through propidium iodide staining. Endothelial and urothelial cell uptake of fluorophore-labeled IPSE was measured. Findings: Injection of S. haematobium eggs into the bladder triggers angiogenesis, enhances leakiness of bladder blood vessels, and drives urothelial hyperplasia. Wild type IPSE, but not NLS-, increases proliferation of endothelial and urothelial cells and skews urothelial cells towards S phase. Finally, IPSE is internalized by both endothelial and urothelial cells. Interpretation: IPSE drives endothelial and urothelial proliferation, which may depend on internalization of the molecule. The urothelial effects of IPSE depend upon its NLS. Thus, IPSE is a candidate pro-carcinogenic molecule of S. haematobium. Summary Schistosoma haematobium acts as a bladder carcinogen through unclear mechanisms. The S. haematobium homolog of IPSE, a secreted schistosome egg immunomodulatory molecule, enhances angiogenesis and urothelial proliferation, hallmarks of pre-carcinogenesis, suggesting IPSE is a key pro-oncogenic molecule of S. haematobium.

Comprehensive genomic profiling enables genomic biomarker detection in advanced solid tumors. Here, to evaluate the utility of circulating tumor DNA (ctDNA) genotyping, we compare trial enrollment using ctDNA sequencing in 1,687 patients with advanced gastrointestinal (GI) cancer in SCRUM-Japan GOZILA (no. UMIN000016343), an observational ctDNA-based screening study, to enrollment using tumor tissue sequencing in the same centers and network (GI-SCREEN, 5,621 patients). ctDNA genotyping significantly shortened the screening duration (11 versus 33 days, P

Clinical studies support the efficacy of programmed cell death 1 (PD-1) targeted therapy in a subset of patients with metastatic gastric cancer (mGC). With the goal of identifying determinants of response, we performed molecular characterization of tissues and circulating tumor DNA (ctDNA) from 61 patients with mGC who were treated with pembrolizumab as salvage treatment in a prospective phase 2 clinical trial. In patients with microsatellite instability-high and Epstein–Barr virus-positive tumors, which are mutually exclusive, dramatic responses to pembrolizumab were observed (overall response rate (ORR) 85.7% in microsatellite instability-high mGC and ORR 100% in Epstein–Barr virus-positive mGC). For the 55 patients for whom programmed death-ligand 1 (PD-L1) combined positive score positivity was available (combined positive score cut-off value ≥1%), ORR was significantly higher in PD-L1(+) gastric cancer when compared to PD-L1(−) tumors (50.0% versus 0.0%, P value

Chemotherapy-induced hemorrhagic cystitis (CHC) can be difficult to manage. Prior work suggests that IL-4 alleviates ifosfamide-induced hemorrhagic cystitis (IHC), but systemically administered IL-4 causes significant side effects. We hypothesized that the Schistosoma hematobium homolog of IL-4-inducing principle from Schistosoma mansoni eggs (H-IPSE), would reduce IHC and associated bladder pathology. IPSE binds IgE on basophils and mast cells, triggering IL-4 secretion by these cells. IPSE is also an "infiltrin," translocating into the host nucleus to modulate gene transcription. Mice were administered IL-4, H-IPSE protein or its nuclear localization sequence (NLS) mutant, with or without neutralizing anti-IL-4 antibody, or 2-mercaptoethane sulfonate sodium (MESNA; a drug used to prevent IHC), followed by ifosfamide. Bladder tissue damage and hemoglobin content were measured. Spontaneous and evoked pain, urinary frequency, and bladdergene expression analysis were assessed. Pain behaviors were interpreted in a blinded fashion. One dose of H-IPSE was superior to MESNA and IL-4 in suppressing bladder hemorrhage in an IL-4-dependent fashion and comparable with MESNA in dampening ifosfamide-triggered pain behaviors in an NLS-dependent manner. H-IPSE also accelerated urothelial repair following IHC. Our work represents the first therapeutic exploitation of a uropathogen-derived host modulatory molecule in a clinically relevant bladder disease model and indicates that IPSE may be an alternative to MESNA for mitigating CHC.-Mbanefo, E. C., Le, L., Pennington, L. F., Odegaard, J. I., Jardetzky, T. S., Alouffi, A., Falcone, F. H., Hsieh, M. H. Therapeutic exploitation of IPSE, a urogenital parasite-derived host modulatory protein, for chemotherapy-induced hemorrhagic cystitis.

Purpose: Plasma cell-free DNA (cfDNA) analysis is increasingly used clinically for cancer genotyping, but may lead to incidental identification of germline-risk alleles. We studied EGFR T790M mutations in non–small cell lung cancer (NSCLC) toward the aim of discriminating germline and cancer-derived variants within cfDNA. Experimental Design: Patients with EGFR-mutant NSCLC, some with known germline EGFR T790M, underwent plasma genotyping. Separately, deidentified genomic data and buffy coat specimens from a clinical plasma next-generation sequencing (NGS) laboratory were reviewed and tested. Results: In patients with germline T790M mutations, the T790M allelic fraction (AF) in cfDNA approximates 50%, higher than that of EGFR driver mutations. Review of plasma NGS results reveals three groups of variants: a low-AF tumor group, a heterozygous group (∼50% AF), and a homozygous group (∼100% AF). As the EGFR driver mutation AF increases, the distribution of the heterozygous group changes, suggesting increased copy number variation from increased tumor content. Excluding cases with high copy number variation, mutations can be differentiated into somatic variants and incidentally identified germline variants. We then developed a bioinformatic algorithm to distinguish germline and somatic mutations; blinded validation in 21 cases confirmed a 100% positive predictive value for predicting germline T790M. Querying a database of 31,414 patients with plasma NGS, we identified 48 with germline T790M, 43 with nonsquamous NSCLC (P < 0.0001). Conclusions: With appropriate bioinformatics, plasma genotyping can accurately predict the presence of incidentally detected germline risk alleles. This finding in patients indicates a need for genetic counseling and confirmatory germline testing. Clin Cancer Res; 23(23); 7351–9. ©2017 AACR.

Introduction The phase 2 CodeBreaK 100 trial evaluated the first-in-class KRASG12C inhibitor, sotorasib, in pts with advanced NSCLC. Pts were enrolled based on the positive KRAS p.G12C status, as determined using tissue-based Qiagen Therascreen KRAS RGQ PCR kit test. Guardant360 (G360) CDx utilizes circulating cfDNA from plasma for sequencing and has been approved by FDA as a companion diagnostic to detect genomic mutations in pts with solid tumors. In this study, we used data from the trial to compare the efficacy of sotorasib in pts positive for KRAS p.G12C by cfDNA with that in the overall population identified by tissue. The concordance between the two assays was evaluated. Methods Key inclusion criteria: centrally confirmed KRAS p.G12C and progression on prior therapies. Primary endpoint was objective response rate (ORR) assessed by central review. Pts who had adequate pretreatment plasma sample were included in the validation. Due to the lack of KRAS p.G12C-negative tissue specimens from the trial population, paired blood and tissue samples from an additional NSCLC cohort of pts were sourced for the concordance study. Results Overall, 126 pts were enrolled based on tissue result. 124 were evaluable for efficacy per central review. 77 of 107 evaluable pts eligible for G360 testing tested positive for KRAS p.G12C. The ORR was 37.1% in pts positive by tissue and 36.4% in those positive by cfDNA (Table). In 189 pts eligible for concordance study, the overall concordance was 81.5%, with positive and negative % agreements of 70.1% and 100.0%, respectively. Conclusions In the phase 2 CodeBreaK 100 trial of sotorasib, efficacy in pts positive for KRAS p.G12C by the plasma-based G360 CDx was comparable to that in the overall population identified by tissue testing, with high concordance demonstrated between the two assays and a negative % agreement of 100%. Plasma cfDNA may be used to identify pts who may benefit from sotorasib. cfDNA by G360 CDx N=77Tissue by Therascreen N=124Best overall response - n (%)Complete response0 (0.0)3 (2.4)Partial response28 (36.4)43 (34.7)Stable disease32 (41.6)54 (43.5)Progressive disease13 (16.9)20 (16.1)Not evaluable/Not done4 (5.2)4 (3.2)Objective response rate - % (95% Cl)36.4 (25.7, 48.1)37.1 (28.6, 46.2) Citation Format: Joshua M. Bauml, Bob T. Li, Vamsidhar Velcheti, Ramaswamy Govindan, Alessandra Curioni Fontecedro, Christophe Dooms, Toshiaki Takahashi, Andrew W. Duda, Justin Odegaard, Fernando Cruz-Guilloty, Liming Jin, Ying Zhang, Ferdinandos Skoulidis. Clinical validation of plasma cell-free DNA (cfDNA) sequencing in the phase 2 trial of sotorasib in patients (pts) with KRAS p.G12C mutated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT181.

Background: NTRK rearrangements, though rare in common cancers, are clinically actionable targets with two FDA-approved drugs for pan-cancer indications, larotrectinib and entrectinib. Fusion detection from ctDNA provides an opportunity to facilitate screening for this biomarker, though technical challenges such as heterogeneity in fusion partners remain. To address this, we developed an assembly-based de novo fusion algorithm that does not rely on a fixed set of partner genes and applied it to >18,000 clinical samples to detect NTRK1 fusions. Methods: A cohort of 18,867 patients across multiple cancer types (lung adenocarcinoma, breast cancer, and colorectal adenocarcinoma), plus 276 healthy control samples were previously tested with Guardant360(R), a CLIA-validated 74-gene cfDNA NGS-based assay. The median unique molecule coverage was approximately 3,000 molecules sequenced to 15,000x read depth. Samples were reanalyzed in silico using the de novo fusion algorithm: in brief, reads aligned to candidate fusion breakpoints were assembled into de Bruijn graphs. Resulting contigs were aligned to the reference and analytical filters were applied to achieve high specificity. Results: NTRK1 fusions were detected in 0.13% of patients, a similar prevalence to what has been previously described in tissue (Rosen et al., 2020). No fusions were identified in 276 healthy control samples. When examining intergenic fusions, 53% of fusions included previously characterized partner genes (TPM3, TPR, LMNA, and TP53), while the remaining fusions contained novel partners.The majority of NTRK1 fusion partners (76%) were detected only once, consistent with previous studies. KRAS G12D and BRAF V600E were detected in 17% and 13% of NTRK1 fusion-positive patients, respectively, and occurred predominantly in the colorectal cohort; most variants (60%) were subclonal to the NTRK1 fusion, and have been previously shown to be associated with resistance to TRK inhibitors through downstream activation of the MAPK pathway. The on-target resistance variant NTRK1 G595R was detected in 8% of patients positive for NTRK1 fusions, all of which were subclonal to the fusion. EGFR L858R was detected in 13% of NTRK1 fusion-positive patients, all of which were clonal to the fusion and in the lung cohort, in accordance with previous studies showing NTRK1 fusions as a resistance mechanism to TKIs in EGFR-positive NSCLC (Xia et al., 2020). Conclusions: NTRK1 fusions were detected in cfDNA at a similar prevalence to tissue NGS, demonstrating high sensitivity of plasma-based assays to detect these fusions. NTRK1 fusion partners were diverse, with the majority of partner genes observed only once across the cohort. Both clonal and subclonal NTRK1 rearrangements were detected, affirming that this biomarker can emerge as an oncogenic driver or as a mechanism of resistance. Citation Format: Arielle Yablonovitch, Sante Gnerre, Jennifer Yen, Scott Shell, Elena Helman, Stephen Fairclough, Rebecca J. Nagy, Justin Odegaard, Darya Chudova, AmirAli Talasaz. NTRK1 fusion detection from clinical cfDNA NGS using a de novo fusion caller [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 537.

Background: Molecular response (MR) estimated as a change in circulating tumor (ctDNA) load between an early on treatment sample (usually 2-9 weeks post treatment start) and pre-treatment baseline has been shown to predict patient response and outcomes across solid tumors and therapy types in many retrospective studies. There is no consensus, however, regarding the best method for assessing molecular response. Therefore, we aimed to assess several molecular response calculations and determine the optimal method for predicting outcomes in individual advanced cancer patients. Method: Aggregate results of > 4,000 patient sample pairs (3-10 weeks apart), > 1000 patient sample technical replicates, > 100 contrived sample dilutions, and in silico simulations were analyzed with Guardant360TM or GuardantOMNITM. Baseline and on-treatment paired patient samples were collected from advanced cancer patients with over 12 tumor types, including lung, colon, and breast. MR calculations included variant allele fractions (VAFs) of somatic SNVs, indels and fusions. Methods including Ratio treatment/baseline (R) of Maximum VAF (maxVAF), Ratio of Mean VAF (mVAF), and Mean of VAF Ratios were compared, with consideration of VAF precision. Analytical accuracy, reproducibility and limit of detection (LoD) were assessed. Results: Comparison of methods for calculating net change in ctDNA load on > 1500 sample pairs showed high correlation (ρ ranged from 0.93 to 0.98) and categorical agreement split by the median (93%). Therefore selecting an optimal method based on outcome prediction would require prohibitively large patient cohorts. Analytical evaluation and in silico simulations can predict the behavior of each method. Simulations of changes in tumor fraction of real pre-treatment samples found that RmVAF or RmaxVAF are more accurate than mVAFR, which can be skewed by low VAF ratios. Almost 25% of sample pairs have a tumor driver or resistance mutation that is not the maxVAF, suggesting tumor dynamics are better captured by mVAF than maxVAF. Newly-detected on-treatment variants can be an important signal of rising ctDNA levels, impacting MR in approximately 2% of sample pairs. Importantly, MR accuracy for all methods decreases as maxVAF approaches or falls below the variant LoD, due to both stochastic detection and higher CV of variants at low VAF. Thus the assay variant LoD is a key determinant of the fraction of patients who can receive MR evaluation. Technical replicates identified the variant criteria at which a 50% change in tumor fraction differs significantly from technical variation, and could define analytical reporting limits. Conclusions: Comparison of MR methods in a large set of patient samples and simulations supports RmVAF with inclusion of newly-detected mutations. Clinical validation of these methods will support patient-specific MR prediction of outcomes. Citation Format: Allysia J. Mak, Katie J. Quinn, Carin Espenschied, Kyle Chang, Han-Yu Chuang, Elena Helman, Darya Chudova, Justin Odegaard, Becky Nagy, AmirAli Talasaz. Comparison of molecular response calculations for prediction of patient outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 401.

3555 Background: HER2 amp occurs in 1-4% of mCRC pts. Two single arm phase 2 studies, HERACLES and MyPathway, showed efficacy for dual HER2-targeted therapy in pts with RAS wild type ( RAS wt) mCRC with HER2 amp detected in tumor tissue; however, efficacy for pts prospectively enrolled with HER2 amp identified in ctDNA is unknown. Furthermore, the efficacy of real-world non-HER2-targeted SOC for HER2 amplified RASwt mCRC pts is not clear. Methods: We conducted a phase 2 trial to evaluate the efficacy of pertuzumab (P) plus trastuzumab (T) in RASwt mCRC pts with HER2 amp centrally confirmed by tissue (IHC and/or FISH) and/or ctDNA (Guardant360) who had progressed on SOC including EGFR blockade. Pts received intravenous P (840 mg loading dose followed by 420 mg) and T (8 mg/kg loading dose followed by 6 mg/kg) every 3 weeks. The primary endpoint was confirmed objective response rate (ORR) by investigator assessment, analyzed for two primary populations: pts with HER2 amp in tissue (tissue+) or in ctDNA (ctDNA+). Efficacy of real-world non-HER2-targeted SOC for HER2 amplified RASwt mCRC pts was prospectively assessed in a concurrent registry: the SCRUM-Japan registry. Results: Among 75 pts screened, concordance of HER2 amp between tissue and ctDNA was 83%. The primary endpoint was met in each cohort of TRIUMPH, with confirmed ORR of 30% (95% CI 14-50%) in 27 tissue+ pts and 28% (12-49%) in 25 ctDNA+ pts. In contrast, ORR in first salvage SOC after EGFR blockade was 0% (0.0-24.7%) in the real-world cohort. Median progression free and overall survival were 4.0 months (1.4-5.6) and 10.1 months (4.5-16.5) in the tissue+ pts and 3.1 months (1.4-5.6) and 8.8 months (4.3-12.9) in the ctDNA+ pts. One pt withdrew due to an adverse event (grade 3 decreased ejection fraction), but no treatment related deaths occurred. In exploratory analyses, pts without ctDNA mutations of RAS/ BRAFV600/ PIK3CA/ HER2 were more likely to respond to P+T than those with a ctDNA mutation in at least one of these genes (ORR 44% vs. 0% in tissue+ and 37% vs. 0% in ctDNA+). Decreased ctDNA fraction and HER2 plasma copy number at 3 weeks after treatment initiation corresponded to P+T response. At least one actionable alteration emerged after progression in 16 (62%) of 26 pts with ctDNA results at both baseline and progression. Among 5 pts who achieved response and had ctDNA results at both time points, 4 pts acquired actionable alteration at progression. Conclusions: We demonstrate promising efficacy and safety of P+T for RASwt mCRC pts with HER2 amp in either tumor tissue or ctDNA. Our results show that complete ctDNA genotyping identifies pts most likely to benefit from dual HER2 blockade and can be used to monitor response and detect actionable resistance biomarkers. Clinical trial information: UMIN000027887 and UMIN000028058.

9027 Background: Somatic genomic testing is recommended by numerous expert guidelines to inform targeted therapy treatment for patients with advanced lung adenocarcinoma (aLUAD). The NILE study was a prospective observational study that demonstrated non-inferiority 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 aLUAD. As the cohort has matured, clinical outcomes data can now be reported. Methods: This prospective, multicenter North American study (NCT03615443) enrolled patients with previously untreated aLUAD who had standard of care (SOC) tissue genotyping performed and concurrent comprehensive cfDNA analysis using the commercially available Guardant360 assay (Guardant Health, Redwood City, CA). After 12 months of study enrollment, objective response rates, disease control rate, and time to treatment data were collected for patients with targetable genomic alterations, as defined by NCCN guidelines, who were treated with physician’s choice of therapy. Results: Among 282 patients on the study, 89 (31.6%) had an actionable biomarker detected by tissue (21.3%) and/or cfDNA (27.3%) analysis. Sixty-one (68.5%) of these patients 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%) achieved a durable response > 6 months; 17 (52%) achieved a durable response > 12 months. Patients responded to targeted therapy regardless of the variant allele frequency of the target alteration. The time to treatment (TtT) was significantly faster for cfDNA-informed biomarker detection as compared to tissue genotyping (median 18 vs 31 days, respectively; p = 0.0008). Conclusions: This is the first prospective community-based study to find that 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 tissue-based targeted therapy clinical outcomes. The NILE study complements and confirms findings in the prospective FLAURA and SLLIP studies, which exclusively enrolled at academic sites. Clinical trial information: NCT03615443.

Purpose: ERBB2 (HER2) amplification is an emerging biomarker in colon cancer, conferring sensitivity to combination anti-HER2 therapy. Measurement of HER2 copy number is typically performed using surgical specimens, but cell-free circulating tumor DNA (ctDNA) analysis may be a noninvasive alternative. We determined the sensitivity of plasma copy number (pCN) for detecting ERBB2 amplifications and whether pCN correlated with tissue-detected copy number. We also assessed response to HER2-targeted therapy based on pCN and suggest a pCN threshold predictive of response. Experimental Design: Forty-eight pretreatment and progression plasma samples from 29 HER2-positive patients in the HERACLES A clinical trial were tested using the Guardant360 cfDNA assay. We correlated ERRB2 pCN with progression-free survival (PFS) and best objective response (BOR) and applied an adjustment method based on tumor DNA shedding using the maximum mutant allele fraction as a surrogate for tumor content to accurately determine the pCN threshold predictive of response. Results: Forty-seven of 48 samples had detectable ctDNA, and 46 of 47 samples were ERBB2-amplified on the basis of cfDNA [2.55–122 copies; 97.9% sensitivity (95% confidence interval, 87.2%–99.8%)]. An adjusted ERBB2 pCN of ≥25.82 copies correlated with BOR and PFS (P = 0.0347). Conclusions: cfDNA is a viable alternative to tissue-based genotyping in the metastatic setting. The cfDNA platform utilized correctly identified 28 of 29 (96.6%) of pretreatment samples as ERBB2-amplified and predicted benefit from HER2-targeted therapy. In this study, an observed pCN of 2.4 and an adjusted pCN of 25.82 copies of ERBB2 are proposed to select patients who will benefit from HER2-targeted therapy.

9553 Background: Genotyping is required to identify cancer patients (pts) eligible for targeted therapy; however, many do not receive biomarker testing, in part due to limitations associated with tissue-only genotyping practices and the growing list of biomarkers recommended to be tested. Liquid biopsy overcomes many of these limitations but is not yet fully adopted. We report here the clinical performance of a comprehensive liquid biopsy test based on next generation sequencing (NGS) of circulating tumor DNA (ctDNA) for the identification of NSCLC patients with EGFR exon 19 deletions (ex19del) or L858R mutations ( EGFRm) or EGFR T790M, eligible for treatment with osimertinib. Methods: 441 (79%) of 556 pts randomized in FLAURA (NCT02296125; first-line osimertinib vs comparator EGFR TKI in EGFRm NSCLC) and 300 (72%) of 419 pts from AURA3 (NCT012151981; osimertinib vs chemotherapy in NSCLC pts with T790M at progression on EGFR TKI) were retrospectively tested with Guardant360 (G360), a 74-gene ctDNA NGS assay assessing single nucleotide variants, insertion-deletions, amplifications, and fusions in genes relevant to targeted therapy selection as well as microsatellite instability. Progression-free survival (PFS) of pts with EGFRm or T790M detected by G360 was compared to pts detected by the cobas EGFR Mutation Test (cobas) using tissue or plasma with an unadjusted cox model. Results: Treatment with osimertinib was associated with a significant PFS benefit relative to control therapy in NSCLC pts with EGFRm (FLAURA) and T790M (AURA3) detected using G360 (Table). Observed clinical benefit for pts with EGFRm or T790M detected by G360 was similar to that for pts with EGFRm or T790M identified by cobas using tissue or plasma specimens. Conclusions: This analysis demonstrates that G360 accurately identifies pts for osimertinib therapy while simultaneously providing comprehensive genotyping for other therapeutic molecular targets. The application of NGS liquid biopsy has the potential to increase rates of pts genotyped and access to precision medicine. [Table: see text]

TPS277 Background: Identifying non-responders to expensive salvage therapies with modest benefits and substantial treatment related adverse events (TRAEs) (e.g. regorafenib/TAS102 in mCRC) is necessary to maximize benefits and limit toxicities. Serial ctDNA sequencing is reliable for tracking tumor dynamics and appears to predict resistance to therapy earlier than radiographic progression. Methods: TACT-D is a randomized study (N = 100) to validate the ability of changes in ctDNA (ΔctDNA) to predict resistance early and in limiting toxicities. We hypothesize that increase in ctDNA (measured by variant allele fraction) at 2 weeks (wk) into treatment can predict resistance earlier than standard radiographic means [at 8-12 wk] and detecting resistance early can enable prompt change in therapy resulting in reduction of TRAEs. Pts with mCRC eligible for either regorafenib/TAS102 are randomized 2:1 to either standard of care (SOC) or ctDNA arm. On SOC arm, treatment is given as per current paradigm i.e. for 8 wk and then restaging. On ctDNA arm, decision to continue therapy is based on ctDNA change between baseline and 2 weeks [ΔctDNA = ctDNA (C1D15 – C1D1)]. Increase in ctDNA triggers early radiographic staging (4 wk). Treatment is continued for disease stability/regression and discontinued for progression. Study has 2 co-primary endpoints: 1) Association of Δ ctDNA and radiographic progression [62 pts on SOC arm, have 94% power (2-sided α 0.05) to detect difference of 95% vs. 58% in progressive disease between pts with increase vs decrease in ctDNA] and 2) Compare proportion of pts experiencing TRAEs within 4 months between study arms [67 in SOC arm and 33 in ctDNA arm have 82% power (2-sided α 0.05) to detect a 30% decrease in toxicity]. Key secondary endpoints include: patient-reported outcomes (MD Anderson Symptom Inventory and PRO-CTCAE), OS, clinical events of special interest (hospitalizations/ER visits/medical interventions such as blood transfusions/IV hydration), clinical trial referral and cost effectiveness. Study is now actively accruing pts (NCT03844620). Funding: MD Anderson Cancer Center, Houston, TX & Guardant Health Inc., Redwood City, CA. Clinical trial information: NCT03844620.

15 Background: Accurate genotyping is mandatory for the management of mCRC pts. Tissue-based testing is still the SoC; however, is not available for all pts. and may be exhausted by serial testing, resulting in incomplete genotyping. We aimed to establish the validity of comprehensive non-invasive ctDNA testing in 1L mCRC pts for whom SoC tissue genotyping was available. Methods: 1L mCRC pts were tested with a comprehensive ctDNA test (Guardant360), a RAS ctDNA test (OncoBeam), and SoC tissue testing at the time of diagnosis. The primary endpoint was NCCN guideline biomarker discovery rate ( KRAS, NRAS, and BRAF mutations, ERBB2 amplification, and microsatellite instability). Results: In 91 evaluable pts, the biomarker discovery rate was 54.9% (50/91) for SoC tissue testing, 59.3% (54/91) for comprehensive ctDNA testing ( p= 0.0318 for non-inferiority vs. SoC), and 42.9% (39/91) for RAS ctDNA testing (inferiority not rejected vs. SoC). Both comprehensive and RAS ctDNA testing showed high positive agreement (85%, 44/52, and 86%, 31/36) and negative agreement (96%, 268/279, and 93%, 93/100) relative to SoC tissue testing at the gene level. Expanding genotyping beyond KRAS codon 12/13 mutations increased biomarker discovery rate by 56% for tissue testing (50/91 vs. 32/91, McNemar’s p< 0.0001) and by 64% for comprehensive ctDNA (54/91 vs. 33/91, McNemar’s p< 0.0001). Turnaround time was significantly shorter for comprehensive ctDNA testing vs. SoC tissue testing (mean 11.7 days vs. 23.0 days, paired T-test p= 0.0002). On retrospective analysis, 92% of biomarker-positive pts would have been identified at 2 weeks using the comprehensive ctDNA test for initial genotyping with reflex to tissue for biomarker-negative pts, whereas initial use of SoC tissue testing would have identified only 85% of positive pts at 4 weeks (Fisher’s exact p< 0.0001). Conclusions: As previously reported for lung cancer, comprehensive ctDNA testing in 1L mCRC identifies at least as many biomarker-positive pts as SoC tissue genotyping with high concordance to tissue and in half the turnaround time.

Ifosfamide and other oxazaphosphorines can result in hemorrhagic cystitis, a constellation of complications caused by acrolein metabolites. We previously showed that a single dose of IPSE, a schistosome-derived host modulatory protein, can ameliorate ifosfamide-related cystitis; however, the exact mechanisms underlying this urotoxic effect and its prevention are not fully understood. To provide insights into IPSE’s protective mechanism, we undertook transcriptional profiling of bladders from ifosfamide-treated mice, with or without IPSE pretreatment. Following ifosfamide challenge, there was upregulation of a range of pro-inflammatory genes. The pro-inflammatory pathway involving the IL-1β, TNFαand IL-6 triad via NFκB and STAT3 signaling pathways was identified as the key driver of inflammation. The NRF2-mediated oxidative stress response pathway, which regulates bothHmox1-mediated heme homoeostasis and expression of antioxidant enzymes, was highly activated. Anti-inflammatory and cellular proliferation cascades implicated in tissue repair, namely Wnt, Hedgehog and PPAR pathways, were downregulated. IPSE administration before ifosfamide injection resulted in significant downregulation of major proinflammatory pathways including the triad of IL-1β, TNFαand IL-6 pathways, the interferon signaling pathway, and less apparent reduction in oxidative stress responses. Taken together, we have identified signatures of acute phase inflammation and oxidative stress responses in the ifosfamide-injured bladder, which are reversed by pretreatment with IPSE, a parasite derived anti-inflammatory molecule. In addition to providing new insights into the underlying mechanism of IPSE’s therapeutic effects, this work has revealed several pathways that could be therapeutically targeted to prevent and treat ifosfamide-induced hemorrhagic cystitis.

Chemotherapy-induced hemorrhagic cystitis is characterized by bladder pain and voiding dysfunction caused by hemorrhage and inflammation. Of currently available therapies, prophylactic 2-mercaptoethanesulfonic acid (MESNA) has limited efficacy and cannot treat pre-existing lesions. Therefore, novel therapeutic options to treat hemorrhagic cystitis are needed. We previously reported that systemic administration of the Schistosomiasis haematobium-derived protein H-IPSEH06 (IL-4-inducing principle from Schistosoma mansonieggs), is superior to 3 doses of MESNA in alleviating hemorrhagic cystitis. Based on prior reports by others on S. mansoni IPSE and additional work by our group, we reasoned that H-IPSEH06 mediates its effects on hemorrhagic cystitis by binding IgE on basophils and inducing IL-4 expression, promoting urothelial proliferation, and translocating to the nucleus to modulate expression of genes implicated in relieving bladder dysfunction. We speculated that local bladder injection of the S. haematobium IPSE ortholog IPSEH03, hereafter called H-IPSEH03, might be more efficacious in preventing hemorrhagic cystitis compared to systemic administration of IPSEH06. We demonstrate herein that H-IPSEH03 is a promising therapeutic for the treatment of voiding dysfunction and bladder pain in hemorrhagic cystitis. Namely, it attenuates ifosfamide-induced increases in bladder wet weight in an IL-4-dependent fashion. H-IPSEH03 relieves hemorrhagic cystitis-associated allodynia. Finally, H-IPSEH03 drives increased urothelial cell proliferation. This indicates that IPSE induces bladder healing mechanisms, which suggests that it may be a novel non-opioid analgesic to treat bladder pain syndromes.

Background Circulating tumor DNA (ctDNA) analysis has been successfully applied to therapy selection and treatment monitoring in advanced cancer patients. However, it is not yet established whether ctDNA can be used clinically for early cancer detection or recurrence prediction in early stage lung cancer patients. Methods We analyzed pre-operative plasma samples from 55 early stage NSCLC patients (stages I-IIIA) using next-generation sequencing assay incorporating somatic and epigenomic analysis, and a bioinformatic classifier to filter non-tumor derived variants. Table: 111P . Cell type Stage Somatic mutation Epigenetic pattern Total number Recurrence+, n (%) Site of recurrence Adenocarcinoma stage 1 ctDNA- methylation- 9 1 (11) Lung n = 17 methylation+ 6 2 (33.3) Stump, bone ctDNA+ 2 1 (50) lung stage 2 ctDNA- methylation- 0 0 (0) n = 2 methylation+ 0 0(0) ctDNA+ 1 1 (100) multiple stage 3 ctDNA- methylation- 0 0 (0) n = 4 methylation+ 2 0 (0) ctDNA+ 2 2 (100) brain, multiple Sqaumous cell carcinoma stage1 ctDNA- methylation- 0 0 (0) n = 7 methylation+ 3 0 (0) ctDNA+ 4 1 (25) multiple stage2 ctDNA- methylation- 0 0 (0) n = 9 methylation+ 0 0 (0) ctDNA+ 9 2 (22.2) multiple, lung stage3 ctDNA- methylation- 0 0 (0) n = 4 methylation+ 1 0 (0) ctDNA+ 3 1 (33.3) Mediastinal LNs Results Somatic mutation analysis alone detected ctDNA in 42% (23/55) of patients, whereas combined mutational and epigenomic analysis detected ctDNA in 67% (37/55). ctDNA detection rate varied by pathological subtypes; using combined approach, ctDNA was detected in all squamous cell carcinoma patients, while only 55% (12/22) in adenocarcinoma (ADC) (p=0.006). Within the ADC subgroup, ctDNA detection rates using the combined approach were dependent on disease stage: 47% (8/17) in stage I, 100% (2/2) in stage II, and 100% (2/2) in stage IIIA. Importantly, within 2 years of follow-up, pre-operative ctDNA status was correlated with tumor recurrence after resection; among 17 stage I ADC patients, three of eight (38%) ctDNA-positive cases showed recurrence, while only one of nine (11%) ctDNA-negative cased did (p=0.29). Interestingly, patients with somatic mutation in their ctDNA have shown higher recurrence rate. Conclusion Utilizing a plasma-only sequencing assay incorporating somatic genomic and epigenomic analysis, ctDNA detection rate in early stage lung cancer (stage I-III) can far outperform the detection rate of somatic sequence variant detection alone. And, the presence of pre-operative ctDNA in patients with early stage lung adenocarcinoma may identify those who are more likely to have disease recurrence. Legal entity responsible for the study: Guradant Health, Inc. Funding Guardant Health, Redwood City, CA, USA. Disclosure I. Kim: Full / Part-time employment, Officer / Board of Directors: Guardant Health. M. Shultz: Officer / Board of Directors: Guardant Health. A. Jaimovich: Officer / Board of Directors: Guardant Health. J. Odegaard: Officer / Board of Directors: Guardant health, Inc. S. Olsen: Officer / Board of Directors: Guardant Health, Inc. A. Talasaz: Officer / Board of Directors: Guardant health. J. Kim: Research grant / Funding (self): Guardant health, Inc. All other authors have declared no conflicts of interest.

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.

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.

Urogenital schistosomiasis, Schistosoma haematobium worm infection, afflicts millions of people with egg-triggered, fibrotic bladder granulomata. Despite the significant global impact of urogenital schistosomiasis, the mechanisms of bladder granulomogenesis and fibrosis are ill defined due to the prior lack of tractable animal models. We combined a mouse model of urogenital schistosomiasis with macrophage-depleting liposomal clodronate (LC) to define how macrophages mediate bladder granulomogenesis and fibrosis. Mice were injected with eggs purified from infected hamsters or vehicle prepared from uninfected hamster tissues (xenoantigen and injection trauma control). Empty liposomes were controls for LC: 1) LC treatment resulted in fewer bladder egg granuloma-infiltrating macrophages, eosinophils, and T and B cells, lower bladder and serum levels of eotaxin, and higher bladder concentrations of IL-1α and chemokines (in a time-dependent fashion), confirming that macrophages orchestrate leukocyte infiltration of the egg-exposed bladder; 2) macrophage-depleted mice exhibited greater weight loss and bladder hemorrhage postegg injection; 3) early LC treatment postegg injection resulted in profound decreases in bladder fibrosis, suggesting differing roles for macrophages in fibrosis over time; and 4) LC treatment also led to egg dose-dependent mortality, indicating that macrophages prevent death from urogenital schistosomiasis. Thus, macrophages are a potential therapeutic target for preventing or treating the bladder sequelae of urogenital schistosomiasis.—Fu, C.-L., Odegaard, J. I., Hsieh, M. H. Macrophages are required for host survival in experimental urogenital schistosomiasis.

Urogenital schistosomiasis is one of the greatest single infectious sources of human morbidity and mortality known. Through a complex cycle of infection, migration and eventual maturation and mating, S. haematobium (the aetiological agent of urogenital schistosomiasis) deposits highly immunogenic eggs within the bladder and other pelvic organs, activating a wide range of immune programs that determine both infection outcome as well as downstream immunopathology. In this review, we discuss the experimental and observational bases for our current understanding of these immune programs, focusing specifically on how the balance of type 1 and type 2 responses governs subsequent immunopathology and clinical outcome.

Understanding how malignancies arise within normal tissues requires identification of the cancer cell of origin and knowledge of the cellular and tissue dynamics of tumour progression. Here we examine bladder cancer in a chemical carcinogenesis model that mimics muscle-invasive human bladder cancer. With no prior bias regarding genetic pathways or cell types, we prospectively mark or ablate cells to show that muscle-invasive bladder carcinomas arise exclusively from Sonic hedgehog (Shh)-expressing stem cells in basal urothelium. These carcinomas arise clonally from a single cell whose progeny aggressively colonize a major portion of the urothelium to generate a lesion with histological features identical to human carcinoma in situ. Shh-expressing basal cells within this precursor lesion become tumour-initiating cells, although Shh expression is lost in subsequent carcinomas. We thus find that invasive carcinoma is initiated from basal urothelial stem cells but that tumour cell phenotype can diverge significantly from that of the cancer cell of origin.

Background: ctDNA has the potential to identify patients (pts) with early stage cancer; however, current assays are challenged by limited sensitivity (~50%), reliance on a single analyte (e.g. somatic mutation detection), and/or the need for tumor tissue or genomic DNA sequencing to interpret ctDNA results. Recent studies have demonstrated that ctDNA can be detected using other biomarkers including DNA methylation. We developed a technology in which both somatic mutations and epigenomic alterations can be analyzed in a single assay. Methods: Using a large database of cell-free DNA (cfDNA) profiles generated from advanced cancer patients, we designed a targeted sequencing assay that detects somatic variants, methylation alterations, and other epigenomic variations at transcription factor binding sites associated with CRC. Total cfDNA was extracted, partitioned based on methylation level, and analyzed. Data were then filtered using a variant classifier to differentiate tumor- from non-tumor-derived alterations without a priori knowledge of tissue or germline sequencing results. A machine learning model was trained on 111 cfDNA samples from 38 late stage and 10 early stage CRC pts and 63 age-matched cancer-free controls. For the independent test set, plasma samples (4-5mL) were collected from 72 pts with stage I-IV CRC prior to and 4 weeks after (N = 50, total of 122 samples) surgical resection. 35 age-matched cancer-free controls were similarly analyzed in the test set. Results: Of the 72 pts, 62.5% were male, and median age at CRC diagnosis was 61.5 years (range 36-85). Stage distribution was 52.8% stage I/II, 40.3% stage III, and 6.9% stage IV. In the 50 pts with post-surgical samples, clinical follow-up was available for 49 (median post-surgery follow-up: 314 days; range 15-472). Utilizing this assay, pre-surgery ctDNA detection rate was 94% (68/72); 97% in stage I/II, 90% in stage III, and 100% in stage IV. Epigenomic analysis significantly enhanced ctDNA detection relative to somatic mutational analysis alone (94% vs. 56%; p Discussion: Utilizing a plasma-only sequencing assay incorporating somatic genomic variant detection, epigenomic analysis, and a bioinformatic classifier to filter non-tumor derived variants, ctDNA detection rate in early stage CRC (I-III) is 94% (63/67; 95% confidence interval 86%;98%) with 94% specificity, far outperforming the detection rate of somatic sequence variant detection alone. Clinical follow-up is ongoing to evaluate post-surgery ctDNA detection rate and disease recurrence. These results have significant implications for the clinical utility of ctDNA in early stage cancer management. Citation Format: Seung-Tae Kim, Victoria M. Raymond, Joon Oh Park, Elena Zotenko, Young Suk Park, Matthew Schultz, Won Ki Kang, Oscar Westesson, Hee-Cheol Kim, Yupeng He, Justin I. Odegaard, Stefanie A. Mortimer, William J. Greenleaf, Ariel Jaimovich, Jeeyun Lee, AmirAli Talasaz. Combined genomic and epigenomic assessment of cell-free circulating tumor DNA (ctDNA) improves assay sensitivity in early-stage colorectal cancer (CRC) [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 916.

3528 Background: Anti-EGFR therapy (tx) in RAS wild-type (wt) metastatic colorectal cancer (mCRC) induces resistance through acquired genomic alterations. We aimed to define such alterations in Nationwide Cancer Genome Screening Project tissue and blood specimens, using next generation sequencing (NGS). Methods: Tumor specimens in patients (pts) with RAS wt mCRC were obtained from SCRUM-Japan GI-SCREEN (tissue) and GOZILA (circulating tumor DNA [ctDNA] from blood). Genomic alterations were compared using the Oncomine Comprehensive Assay (SCRUM) and Guardant360 (GOZILA), before anti-EGFR tx and after progression. Results: 373 total actionable alterations were identified in 71 pts with available matched tissue and ctDNA; 255 (68%) were acquired after anti-EGFR tx progression. Frequently seen acquired oncogenic alterations included KRAS mutations (27%) and amplifications (amps) of EGFR (41%), CDK6 (24%), BRAF (20%), MYC (17%), MET (14%), PIK3CA (11%), FGFR1 (11%), and KRAS (10%). Fusions of RET, ALK, and FGFR3 were newly acquired in 1-4%. Acquired alterations co-arose in multiple pathways, including the cell cycle, PI3K-AKT, and MAPK, although 29% of pts had none. Acquired mutations were less frequently clonal versus primary mutations (p7) adjusted plasma copy numbers (ApCN). Acquired ERBB2 amps were identified in 3 pts (4%) with a median ApCN of 4, one of whom (ApCN=4.2), treated with dual HER2 blockade, progressed after 2 cycles. Conclusions: Our integrated analysis revealed that anti-EGFR tx of pts with RAS WT mCRC led to acquired genomic alterations in multiple oncogenic pathways. Although most acquired alterations were subclonal, a subset of oncogenic alterations had relatively high clonality and ApCN, suggesting potential targets for overcoming acquired resistance to anti-EGFR tx. Early progression in a pt with an ApCN of 4.2 suggests low-level/subclonal acquired alterations may not be effective treatment targets.

e14557 Background: Circulating tumor DNA (ctDNA) analysis has been successfully applied to therapy selection and treatment monitoring in advanced cancer patients. However, it is not yet established whether ctDNA can be used clinically for early cancer detection or predicting tumor recurrence in early stage lung cancer patients. Methods: We analyzed pre-operative plasma samples from 55 early stage NSCLC patients (stages I-IIIA) using next-generation sequencing to detect somatic mutations and differential epigenomics patterns, including methylation signatures. Results: Using somatic mutation analysis alone, ctDNA was detected in 42% (23/55) of patients, whereas combined mutational and epigenomic analysis detected ctDNA in 71%. ctDNA detection rate also varied markedly between lung squamous cell carcinoma (SCC) and adenocarcinoma (ADC);using combined analysis of somatic mutations and epigenomic patterns, ctDNA was detected in all SCC patients, while only 55% of ADC (12/22) were ctDNA-positive (p= 0.006). Within the ADC subgroup, ctDNA detection rates using the combined approach were dependent on disease stage: 47% (8/17) in stage I, 100% (2/2) in stage II, and 100% (2/2) in stage IIIA. Importantly, pre-operative ctDNA status was correlated with tumor recurrence post-resection; three of eight (38%) ctDNA-positive stage I ADC patients recurred within 2 years of resection, while only one of nine (11%) ctDNA-negative stage I ADC patients recurred (p= 0.29). Conclusions: Taken together, we show that the combination of somatic mutation detection and epigenomic analysis outperforms each individual biomarker in the detection of ctDNA in early stage lung cancer. Importantly, we also demonstrate that pre-operative ctDNA detection may identify a high-risk population of early stage lung cancer patients that may benefit from (neo)adjuvant therapy.

The liver is a central organ for the synthesis and storage of nutrients, production of serum proteins and hormones, and breakdown of toxins and metabolites. Because the liver is susceptible to toxin- or pathogen-mediated injury, it maintains a remarkable capacity to regenerate by compensatory growth. Specifically, in response to injury, quiescent hepatocytes enter the cell cycle and undergo DNA replication to promote liver regrowth. Despite the elucidation of a number of regenerative factors, the mechanisms by which liver injury triggers hepatocyte proliferation are incompletely understood. We demonstrate here that eosinophils stimulate liver regeneration after partial hepatectomy and toxin-mediated injury. Liver injury results in rapid recruitment of eosinophils, which secrete IL-4 to promote the proliferation of quiescent hepatocytes. Surprisingly, signaling via the IL-4Rα in macrophages, which have been implicated in tissue repair, is dispensable for hepatocyte proliferation and liver regrowth after injury. Instead, IL-4 exerts its proliferative actions via IL-4Rα in hepatocytes. Our findings thus provide a unique mechanism by which eosinophil-derived IL-4 stimulates hepatocyte proliferation in regenerating liver.

Metabolism and immunity are inextricably linked both to each other and to organism-wide function, allowing mammals to adapt to changes in their internal and external environments. In the modern context of obesogenic diets and lifestyles, however, these adaptive responses can have deleterious consequences. In this Review, we discuss the pleiotropic actions of inflammation and insulin resistance in metabolic homeostasis and disease. An appreciation of the adaptive context in which these responses arose is useful for understanding their pathogenic actions in disease.

Hereditary diffuse gastric cancer is a rare, autosomal dominant hereditary cancer syndrome associated with germline mutations in CDH1 in which 60% - 80% of affected individuals develop advanced diffuse gastric cancer, many as young adults. At clinical presentation, ~90% of these malignancies represent advanced, surgically incurable disease. As such, presymptomatic identification of germline CDH1 mutation carriers followed by early prophylactic total gastrictomy is the sole effective management strategy available. DNA sequence analysis of the CDH1 gene to identify the affected germline allele is the diagnostic standard of care; however, CDH1’s relatively high frequency of polymorphisms and the limited amount of experience available regarding them dictate that many identified variants are, as yet, of unknown clinical significance. Given the dramatic consequences of inappropriately offered or withheld treatment, careful clinical selection of at-risk individuals is critical. To facilitate this, multiple groups have published screening criteria recommendations, and while there is disagreement regarding the optimal diagnostic approach, the most widely-used overlap to a considerable degree.

Background: Numerous studies have shown platelet-rich plasma (PRP) preparations differ with respect to the inclusion of certain blood components, which may affect the host’s cellular response. Hypothesis: This study evaluated the inflammatory effect of Biomet GPS III leukocyte-rich PRP (LR-PRP) versus MTF Cascade leukocyte-poor PRP (LP-PRP) after intratendinous injection in an animal model. The authors anticipated that LR-PRP would incite a greater acute inflammatory response than LP-PRP. Study Design: Controlled laboratory study. Methods: A total of 17 skeletally mature New Zealand White rabbits were tested. In all cases, healthy patellar tendons were treated. In the control animals, one patellar tendon was injected with 2 mL autologous whole blood, and the other was injected with 2 mL sterile saline. Seven total tendons were injected with whole blood, and 7 tendons were injected with saline. In the experimental animals, one patellar tendon was injected with 2 mL LR-PRP, and the other was injected with 2 mL LP-PRP. Ten tendons were injected with LR-PRP, and 10 tendons were injected with LP-PRP. Animals were euthanized at 5 or 14 days after injection. Tendons were harvested and stained using hematoxylin and eosin and scored semi-quantitatively for total white blood cells (WBCs), mononuclear cells (macrophages and lymphocytes), polymorphonuclear cells (PMNs), vascularity, fiber structure, and fibrosis. Results: At 5 days after injection, tendons treated with LR-PRP had significantly greater overall tendon scores (6.3 ± 1.79 vs 1.8 ± 1.64, P = .012), as well as mean scores for fiber structure (1.4 ± 0.22 vs 0.50 ± 0.50, P = .012), denoting disrupted composition, total WBCs (1.1 ± 0.89 vs 0.10 ± 0.22, P = .014), mononuclear cells (macrophages and lymphocytes) (0.80 ± 0.45 vs 0.10 ± 0.22, P = .014), vascularity (1.7 ± 0.27 vs 0.80 ± 0.16, P = .008), and fibrosis (1.0 ± 0.35 vs 0.3 ± 0.45, P = .037) compared with tendons treated with LP-PRP. Otherwise, there were no significant differences in mononuclear cells ( P = .590), PMN cells ( P = 1.00), total WBCs ( P = .811), vascularity ( P = .650), or total tendon score ( P = .596) in any of the treatment groups at 14 days. Conclusion: Compared with leukocyte-poor Cascade PRP, leukocyte-rich GPS III PRP causes a significantly greater acute inflammatory response at 5 days after injection. There is no significant difference in the inflammatory response or cellularity regardless of the injection type at 14 days after intratendinous injection. Clinical Relevance: Platelet-rich plasma injections are frequently prepared using commercial systems and are administered for clinical treatment of chronic tendinopathy. It is important to characterize the cellular responses elucidated by different injection preparations to further understand their effect on tissue healing and aid clinical decision making. Future investigations are necessary to apply these findings to the clinical setting.

Purpose To evaluate the safety and effectiveness of the excimer laser sheath technique for removing embedded inferior vena cava (IVC) filters. Materials and Methods Over 12 months, 25 consecutive patients undergoing attempted IVC filter retrieval with a laser-assisted sheath technique were prospectively enrolled into an institutional review board–approved study registry. There were 10 men and 15 women (mean age 50 years, range 20–76 years); 18 (72%) of 25 patients were referred from an outside hospital. Indications for retrieval included symptomatic filter-related acute caval thrombosis (with or without acute pulmonary embolism), chronic IVC occlusion, and bowel penetration. Retrieval was also performed to remove risks from prolonged implantation and potentially to eliminate need for lifelong anticoagulation. After failure of standard methods, controlled photothermal ablation of filter-adherent tissue with a Spectranetics laser sheath and CVX-300 laser system was performed. All patients were evaluated with cavography, and specimens were sent for histologic analysis. Results Laser-assisted retrieval was successful in 24 (96%) of 25 patients as follows: 11 Gunther Tulip (mean 375 days, range 127–882 days), 4 Celect (mean 387 days, range 332–440 days), 2 Option (mean 215 days, range 100–330 days), 4 OPTEASE (mean 387 days, range 71–749 days; 1 failed 188 days), 2 TRAPEASE (mean 871 days, range 187–1,555 days), and 2 Greenfield (mean 12.8 years, range 7.2–18.3 years). There was one (4%) major complication (acute thrombus, treated with thrombolysis), three (12%) minor complications (small extravasation, self-limited), and one adverse event (coagulopathic retroperitoneal hemorrhage) at follow-up (mean 126 days, range 13–302 days). Photothermal ablation of filter-adherent tissue was histologically confirmed in 23 (92%) of 25 patients. Conclusions The laser-assisted sheath technique appears to be a safe and effective tool for retrieving embedded IVC filters, including permanent types, with implantation ranging from months to > 18 years.

Obesity and its attendant metabolic disorders represent the great public health challenge of our time. Recent evidence suggests that onset of inflammation in metabolic tissues pathogenically links obesity to insulin resistance and type 2 diabetes. In this review, we briefly summarize the extant literature, paying special attention to the central role of the tissue-associated macrophage in the initiation of metabolic inflammation. We argue that rather than representing simple inflammatory disease, obesity and metabolic syndrome represent derangements in macrophage activation with concomitant loss of metabolic coordination. As such, the sequelae of obesity are as much products of the loss of positive macrophage influences as they are of the presence of deleterious inflammation. The therapeutic implications of this conclusion are profound because they suggest that pharmacologic targeting of macrophage activation, rather than simply inflammation, might be efficacious in treating this global epidemic.

Objective High altitude headache (HAH) is the most common neurological complaint at altitude and the defining component of acute mountain sickness (AMS). However, there is a paucity of literature concerning its prevention. Toward this end, we initiated a prospective, double-blind, randomized, placebo-controlled trial in the Nepal Himalaya designed to compare the effectiveness of ibuprofen and acetazolamide for the prevention of HAH. Methods Three hundred forty-three healthy western trekkers were recruited at altitudes of 4280 m and 4358 m and assigned to receive ibuprofen 600 mg, acetazolamide 85 mg, or placebo 3 times daily before continued ascent to 4928 m. Outcome measures included headache incidence and severity, AMS incidence and severity on the Lake Louise AMS Questionnaire (LLQ), and visual analog scale (VAS). Results Two hundred sixty-five of 343 subjects completed the trial. HAH incidence was similar when treated with acetazolamide (27.1%) or ibuprofen (27.5%; P = .95), and both agents were significantly more effective than placebo (45.3%; P = .01). AMS incidence was similar when treated with acetazolamide (18.8%) or ibuprofen (13.7%; P = .34), and both agents were significantly more effective than placebo (28.6%; P = .03). In fully compliant participants, moderate or severe headache incidence was similar when treated with acetazolamide (3.8%) or ibuprofen (4.7%; P = .79), and both agents were significantly more effective than placebo (13.5%; P = .03). Conclusions Ibuprofen and acetazolamide were similarly effective in preventing HAH. Ibuprofen was similar to acetazolamide in preventing symptoms of AMS, an interesting finding that implies a potentially new approach to prevention of cerebral forms of acute altitude illness.

Background: Translational research and enrollment in clinical trials are limited by the rarity of individual mutations and lack of sufficient tissue for comprehensive testing. To address these limitations, we developed GuardantOMNI (OMNI), a highly sensitive 500-gene cfDNA sequencing test requiring as little as 2 mL of plasma and designed for broad genomic detection of somatic single-nucleotide variants (SNVs) and small indels in 497 genes, copy number amplifications (CNAs) in 106 genes, and fusions in 21 genes. Additionally, the OMNI panel enables assessment of tumor mutational burden (TMB), and DNA damage and mismatch repair, with coverage of over 30 genes associated with the DDR pathway. Here we present the first analytical validation study of OMNI. Methods: Analytical performance was assessed as per Nex-StoCT Working Group guidelines using precharacterized cell lines and healthy normal donor-derived samples. Qualitative and quantitative orthogonal confirmation was provided by exome sequencing, microarrays, and data from published compendia. Results: Seventy-three validation and 150 development plasma samples were processed for this study, using both 5ng and 30ng cfDNA input levels, and all samples passed sequencing QC metrics established prior to testing. Reportable ranges for SNVs were ≥0.04% variant allele fraction (VAF), ≥0.02% for indels, ≥2 supporting molecules for fusions, and ≥2.18 copies for CNAs. Cell line-based dilution studies demonstrated 95% limits of detection (LoD) of 0.24-0.6% VAF for SNVs (depending on known cancer association), 0.4-0.8% for non-homopolymeric indels (depending on clinical relevance), 0.1-0.2% for fusions, and 2.2-2.9 copies for 90% of CNA genes targeted. Comparison of diluted cell line and healthy donor samples to orthogonal sequencing and published genotype data demonstrated accuracies of 98.7% for SNVs, 97.2% for indels, and 100% for CNAs and fusions across the reportable range. The analytical false-positive rate per sample measured across 24 healthy donors was 0.25 for SNVs, 0.04 for indels, and 0 for CNAs and fusions, with positive predictive values (PPVs) of 97.5% for SNVs, 98% for indels, and 100% for CNAs and fusions. Quantitative correlation of allele fraction with confirmatory methods was high (r2 > 0.99). Conclusions: To our knowledge, OMNI is the largest comprehensive ctDNA cancer gene panel available. It detects alterations in genes under study in over 98% of current clinical trials with sensitivity, specificity, and accuracy similar to currently available targeted ctDNA sequencing tests. OMNI has the potential to accelerate clinical trial enrollment, research and discovery with a single, noninvasive blood sample. Citation Format: Elena Helman, Carlo Artieri, James V. Vowles, Jennifer Yen, Tracy Nance, Marcin Sikora, Joshua Gourneau, Mohit Goel, Stefanie Mortimer, Darya Chudova, Justin Odegaard, Richard B. Lanman, AmirAli Talasaz. Analytical validation of a comprehensive 500-gene ctDNA panel designed for immuno-oncology and DNA damage research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5603.

3506Background: The rate of HER2 (ERBB2) copy number amplification (CNA) in metastatic colorectal cancer (mCRC) ranges from 2-13%. HERACLES, a phase II trial of trastuzumab and lapatinib (T+L) in H...

12044Background: ctDNA in post-op CRC pts correlates with molecular residual disease and may be useful to guide adjuvant therapy. However, initial studies employed clinically impractical assays ind...

Chronic inflammation is now recognized as a key step in the pathogenesis of obesity-induced insulin resistance and type 2 diabetes mellitus. This low-grade inflammation is mediated by the inflammatory (classical) activation of recruited and resident macrophages that populate metabolic tissues, including adipose tissue and liver. These findings have led to the concept that infiltration and activation of adipose tissue macrophages is causally linked to obesity-induced insulin resistance. Studies have shown, however, that alternatively activated macrophages taking residence in adipose tissue and liver perform beneficial functions in obesity-induced metabolic disease. By attenuating tissue inflammation and increasing oxidative metabolism in liver and skeletal muscle, alternatively activated macrophages have lessened insulin resistance in obese mice. The discovery that distinct subsets of macrophages are involved in the promotion or attenuation of insulin resistance suggests that pathways controlling macrophage activation can potentially be targeted to treat these co-morbidities of obesity. Thus, this Review focuses on the stimuli and mechanisms that control classical and alternative activation of tissue macrophages, and how these macrophage activation programs modulate insulin action in peripheral tissues. The functional importance of macrophage activation is further discussed in the context of host defense to highlight the crosstalk between innate immunity and metabolism.

Bladder cancers arise from transformed urothelial cells that line the bladder. These cancers are urothelial or squamous cell carcinomas or, more rarely, additional histologic variants such as adenocarcinoma. The most important bladder cancer risk factors worldwide are arguably smoking and urogenital schistosomiasis. The parasitic Schistosoma haematobium worm causes urogenital schistosomiasis in approximately 112 million people, primarily in sub-Saharan Africa and the Middle East [1]. During infection, S. haematobium worms lay highly inflammatory eggs in the bladder wall. This inflammation is thought to promote carcinogenesis through unclear mechanisms. People with chronic urogenital schistosomiasis exhibit increased risk and earlier onset of bladder cancer (up to two decades earlier), with a predominance of squamous cell carcinoma [2]. Consequently, S. haematobium has been categorized as a Group I carcinogen (“carcinogenic to humans”) by the International Agency on Research on Cancer of the World Health Organization [3].

A multidisciplinary approach with video-assisted thoracoscopic surgery and laparoscopy was used to manage a very rare case of bilateral catamenial pneumothoraces and pelvic endometriosis., Introduction: Endometriosis of the lung and the diaphragm is rare. Patients may present with symptoms such as shortness of breath, chest pain, and shoulder pain or they may be asymptomatic. Of note, there have been few reports of bilateral catamenial disease, and no reports, to our knowledge, of bilateral pathology proven pulmonary parenchymal endometriosis. Case: A 43-year-old with stage IV endometriosis and large leiomyoma underwent a laparoscopic hysterectomy and treatment of endometrial lesions in 2005. In March and April of 2011, she presented with bilateral pneumothoraces. She subsequently underwent video-assisted thoracoscopy as well as resection and fulguration of bilateral lung and diaphragmatic endometriosis. Pathology confirmed endometrial implants in the lung parenchyma bilaterally. Conclusion: Catamenial pneumothorax is the most common presentation of thoracic endometriosis. However, bilateral catamenial pneumothoraces are rare. To the best of our knowledge, this case reflects the first report of pathology proven bilateral lung and diaphragm endometriosis.

Background: Non-invasive genotyping of cell-free DNA (cfDNA) provides physicians with the ability to identify genomic alterations, evaluate response to treatment, and uncover potential mechanisms of resistance. Here, we analyze the concordance of next-generation sequencing (NGS) and droplet digital PCR (ddPCR) in detecting and quantifying actionable mutations from cfDNA of lung, colorectal, and breast cancer patients. Methods: We studied a cohort of 221 advanced cancer cfDNA samples with completed Guardant360 digital sequencing results. Variant allele frequencies (% AFs) ranged from less than 0.2% to greater than 90% for common driver and resistance mutations. Blinded to plasma NGS results, we performed ddPCR using our validated assays for EGFR L858R, exon19 del, T790M (113 presumed lung cases); BRAF V600E, KRAS G12X (77 colorectal cases); ESR1 D538G, Y537C and PIK3CA H1047R (31 breast cases). Following unblinding, qualitative and quantitative accuracy were compared. Results: Qualitative results (Table 1) show a high concordance between NGS and ddPCR, with 98.3% (576/586) of all assays run being concordant. 9/22 of samples that had an AF below 0.4% were discordant, while only one sample above 0.4% AF was discordant. AFs calculated by NGS and ddPCR were strongly correlated by linear regression (R between 0.951 and 0.999). Notably, the slope of the ddPCR AF versus NGS AF graph for EGFR ex19del was 0.725, which improved to 0.95 with the adoption of an alternate calling method. Conclusions: This interlaboratory comparison of actionable mutations in cfDNA from lung, colorectal, and breast cancers by orthogonal genotyping platforms demonstrates a substantial concordance in detection and quantification between NGS and ddPCR. Our cross-platform study establishes the feasibility of standardization of qualitative and quantitative measurements of cfDNA-detected alterations. Table 1LungEGFR L858REGFR Ex19 delEGFR T790M(n=113)G360+G360-G360+G360-G360+G360-ddPCR+580510530ddPCR-055458456BreastESR1 Y537CESR1 D538GPIK3CA H1047R(n=31)G360+G360-G360+G360-G360+G360-ddPCR+1022020ddPCR-03009029ColorectalBRAF V600EKRAS G12X(n=78)G360+G360-G360+G360-ddPCR+220571ddPCR-055118 Citation Format: Bryan C. Ulrich, Rebecca J. Nagy, Justin I. Odegaard, Richard B. Lanman, Geoffrey R. Oxnard, Cloud P. Paweletz. Cross-platform detection and quantification of actionable mutations in cell-free DNA shows high concordance and correlation between next-generation sequencing and droplet digital PCR [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 5692. doi:10.1158/1538-7445.AM2017-5692

Guardant360 is a cell-free circulating tumor DNA (ctDNA) test that genotypes all guideline-recommended solid tumor somatic genomic treatment targets from a single non-invasive blood draw. The new version, v2.10, was redesigned to enhance sensitivity and specificity across 73 cancer-related genes. It detects all four major variant classes (single nucleotide variants, SNVs, in all 73 genes; indels in 23 genes; gene amplifications, CNAs, in 18 genes; and fusions in 6 genes). Analytical performance was assessed throughout the reportable range via multiple serial dilution studies of orthogonally-characterized contrived and patient samples. Analytical specificity was assessed by calculating the false positive rate in pre-characterized healthy donor sample mixtures serially diluted. Positive predictive value (PPV) was estimated as a function of allelic fraction/copy number from pre-characterized samples and prevalence-adjusted using a cohort of 2,585 consecutive clinical samples. Confirmation was performed using ddPCR. Analytical specificity was 100% for SNVs, fusions, and CNAs and 96% for indels across 25 defined samples. Relative to Guardant360v2.9, v2.10 demonstrated 20-50% increase in fusion molecule recovery. Retrospective in silico analysis of 2,585 consecutive clinical samples demonstrated a 15% increase in actionable fusion detection, a 6%-15% increase in actionable indel detection (excluding newly reportable indels), and a 3%-6% increase in actionable SNV detection. AlterationsReportable Range95% Limit of DetectionAllelic Fraction / Copy NumberAnalytical SensitivityAllelic Fraction / Copy numberPPVSNVs≥0.04%0.25%≥0.25%>99.9%≥0.25%98.7%0.05–0.25%63.8%99.9%≥0.25%98.4%0.05–0.25%67.8% Guardant360 analytical performance characteristics based on standard cfDNA input (30ng). Analytical sensitivity/limit of detection estimates are provided for clinically actionable variants and may vary by sequence context and cfDNA input. PPV is estimated across entire reportable panel space (PPV was 100% for clinically actionable variants). Conclusion: Guardant360 v2.10 comprehensively detects all adult solid tumor guideline-recommended somatic genomic variants with unparalleled sensitivity, accuracy, and specificity. Citation Format: James Vowles, Justin Odegaard, Stefanie Mortimer, Stephen Fairclough, Marcin Sikora, Diana Abdueva, Reza Mokhtari, Arthur Baca, AmirAli Talasaz. Analytical validation of Guardant360 v2.10 [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 5705. doi:10.1158/1538-7445.AM2017-5705

1514 Background: No studies have yet described incidental detection of germline cancer predisposition mutations using circulating cell-free DNA (cfDNA). Methods: Deidentified cfDNA sequencing data from 10288 advanced cancer patients (pts) undergoing clinical circulating tumor DNA testing (Guardant360, 73 genes) were included in this study. CfDNA was extracted from plasma and quantified. A DNA library was prepared and sequenced to 15,000X average read depth. Using Ingenuity Variant Analysis, point mutations and small indels suspicious for germline origin (allele fraction 40-60%) were classified following American College of Medical Genetics and Genomics guidelines. Results: More than 50 cancer types were studied, including lung (40%), breast (20%), CRC (8%), prostate (6%), and pancreas (3%). Average age was 63.6 years (range:18-95), 42% were male. Of 34,873 putative germline variants identified, 520 (1.5%) were pathogenic or likely pathogenic (PV), 16,939 (49%) were of uncertain significance, and 17,414 (50%) were benign or likely benign. Of the 250 pts (2.4%) with hereditary cancer syndrome gene PVs, 83 were excluded due to high level of somatic tumor burden leaving 167 (1.6%) with putative germline PVs; rates were higher in pts 50 overall (3.3% vs 1.4%, p=0.02) and in breast cancer pts (4.3% vs 1.5%, p=0.03). Conclusions: The observed frequency of incidentally identified putative germline PVs is expectedly lower than the true germline rate; however, these findings illustrate that detection from cfDNA is clinically feasible. Importantly, incidental germline findings could impact oncology treatment planning (e.g. PARP inhibitors for BRCA1/2 mutations) and could benefit families via increased surveillance/primary prevention. Further research is needed to explore how to report potential germline results to clinicians using a systems-based approach. [Table: see text]