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Alpelisib is a selective inhibitor of PI3Kα, shown to improve outcomes for PIK3CA mutant, hormone receptor positive (HR+) metastatic breast cancers (MBC) when combined with antiestrogen therapy. To uncover mechanisms of resistance, we conducted a detailed, longitudinal analysis of tumor and plasma circulating tumor DNA among such patients from a phase I/II trial combining alpelisib with an aromatase inhibitor (AI) (NCT01870505). The trial's primary objective was to establish safety with maculopapular rash emerging as the most common grade 3 adverse event (33%). Among 44 evaluable patients, the observed clinical benefit rate was 52%. Correlating genetic alterations with outcome, we identified loss-of-function PTEN mutations in 25% of patients with resistance. ESR1 activating mutations also expanded in number and allele fraction during treatment and were associated with resistance. These data indicate that genomic alterations that mediate resistance to alpelisib or antiestrogen may promote disease progression and highlight PTEN loss as a recurrent mechanism of resistance to PI3Kα inhibition.

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 VIKTORY (targeted agent eValuation In gastric cancer basket KORea) trial was designed to classify patients with metastatic gastric cancer based on clinical sequencing and focused on eight different biomarker groups (RAS aberration, TP53 mutation, PIK3CA mutation/amplification, MET amplification, MET overexpression, all negative, TSC2 deficient, or RICTOR amplification) to assign patients to one of the 10 associated clinical trials in second-line (2L) treatment. Capivasertib (AKT inhibitor), savolitinib (MET inhibitor), selumetinib (MEK inhibitor), adavosertib (WEE1 inhibitor), and vistusertib (TORC inhibitor) were tested with or without chemotherapy. Seven hundred seventy-two patients with gastric cancer were enrolled, and sequencing was successfully achieved in 715 patients (92.6%). When molecular screening was linked to seamless immediate access to parallel matched trials, 14.7% of patients received biomarker-assigned drug treatment. The biomarker-assigned treatment cohort had encouraging response rates and survival when compared with conventional 2L chemotherapy. Circulating tumor (ctDNA) analysis demonstrated good correlation between high MET copy number by ctDNA and response to savolitinib. Significance: Prospective clinical sequencing revealed that baseline heterogeneity between tumor samples from different patients affected response to biomarker-selected therapies. VIKTORY is the first and largest platform study in gastric cancer and supports both the feasibility of tumor profiling and its clinical utility. This article is highlighted in the In This Issue feature, p. 1325

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

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

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 Parasitic infections can increase susceptibility to bacterial co-infections. This may be true for urogenital schistosomiasis and bacterial urinary tract co-infections (UTI). We previously reported that this co-infection is facilitated by S. haematobium eggs triggering interleukin-4 (IL-4) production and sought to dissect the underlying mechanisms. The interleukin-4-inducing principle from Schistosoma mansoni eggs (IPSE) is one of the most abundant schistosome egg-secreted proteins and binds to IgE on the surface of basophils and mast cells to trigger IL-4 release. IPSE can also translocate into host nuclei using a nuclear localization sequence (NLS) to modulate host transcription. We hypothesized that IPSE is the factor responsible for the ability of S. haematobium eggs to worsen UTI pathogenesis. Methods Mice were intravenously administered a single 25 μg dose of recombinant S. haematobium-derived IPSE, an NLS mutant of IPSE or PBS. Following IPSE exposure, mice were serially weighed and organs analyzed by histology to assess for toxicity. Twenty-four hours after IPSE administration, mice were challenged with the uropathogenic E. coli strain UTI89 by urethral catheterization. Bacterial CFU were measured using urine. Bladders were examined histologically for UTI-triggered pathogenesis and by PCR for antimicrobial peptide and pattern recognition receptor expression. Results Unexpectedly, IPSE administration did not result in significant differences in urine bacterial CFU. However, IPSE administration did lead to a significant reduction in UTI-induced bladder pathogenesis and the expression of anti-microbial peptides in the bladder. Despite the profound effect of IPSE on UTI-triggered bladder pathogenesis and anti-microbial peptide production, mice did not demonstrate systemic ill effects from IPSE exposure. Conclusions Our data show that IPSE may play a major role in S. haematobium-associated urinary tract co-infection, albeit in an unexpected fashion. These findings also indicate that IPSE either works in concert with other IL-4-inducing factors to increase susceptibility of S. haematobium-infected hosts to bacterial co-infection or does not contribute to enhancing vulnerability to this co-infection. Graphical Abstract

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.

The optimal perioperative management of patients who undergo hepatectomy for resectable colorectal liver metastases (CRLM) remains unclear due to the lack of reliable methods to stratify the risk of recurrence. A single-center retrospective study was performed to investigate the impact of preoperative circulating tumor DNA (ctDNA) on survival outcomes of patients who underwent initial hepatectomy for solitary resectable CRLM between January 2005 and December 2017 using the comprehensive genotyping platform Guardant360®. Of 212 patients who underwent initial hepatectomy for solitary resectable CRLM, 40 patients for whom pre-hepatectomy plasma was available underwent ctDNA analysis. Among them, 32 (80%) had at least 1 somatic alteration in their ctDNA, while the other 8 (20%) had no detectable ctDNA. Among the patients with undetectable ctDNA, only one had recurrence and none died during a median follow-up period of 39.0 months. The recurrence-free survival was significantly shorter in patients who were positive for ctDNA than in those who were negative for ctDNA [median, 12.5 months vs not reached (NR); HR, 7.6; P = 0.02]. The overall survival also tended to be shorter in patients who were positive for ctDNA than those who were negative for ctDNA (median, 78.1 months vs NR; P = 0.14; HR not available). In patients undergoing hepatectomy for solitary resectable CRLM, the absence of detectable preoperative ctDNA identified patients with a high chance for a cure. Risk stratification according to preoperative ctDNA analysis may be an effective tool that can improve the perioperative management of these patients.

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

Cell-free DNA (cfDNA) next-generation sequencing has the potential to capture tumor heterogeneity and genomic evolution under treatment pressure in a non-invasive manner. Here, we report the detection of EGFR L792 mutations, a non-covalent mechanism of osimertinib resistance, using Guardant360 cfDNA testing in a patient with metastatic EGFR-mutant non-small cell lung cancer (NSCLC) whose disease progressed on osimertinib. We subsequently analyzed a large cohort of over 1800 additional patient samples harboring an EGFR T790M mutation and identified a concomitant L792 mutation in a total of 22 (1.2%) cases. In vitro functional assays demonstrated that the EGFR L858R/T790M/L792F/H mutations conferred intermediate-level resistance to osimertinib. Further understanding of potential acquired resistance mechanisms to targeted therapy may help inform treatment strategy in EGFR-mutant NSCLC.

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.

Background Viral infections are a major cause of complications and death in solid organ and hematopoietic cell transplantation. Methods We developed a multiplex viral sequencing assay (mVseq) to simultaneously detect 20 transplant-relevant DNA viruses from small clinical samples. The assay uses a single-tube multiplex PCR to amplify highly conserved virus genomic regions without the need for previous virus enrichment or host nucleic acid subtraction. Multiplex sample sequencing was performed using Illumina MiSeq, and reads were aligned to a database of target sequences. Analytical and clinical performance was evaluated using reference viruses spiked into human plasma, as well as patient plasma and nonplasma samples, including bronchoalveolar lavage fluid, cerebrospinal fluid, urine, and tissue from immunocompromised transplant recipients. Results For the virus spike-in samples, mVseq's analytical sensitivity and dynamic range were similar to quantitative PCR (qPCR). In clinical specimens, mVseq showed substantial agreement with single-target qPCR (92%; κ statistic, 0.77; 259 of 282 viral tests); however, clinical sensitivity was reduced (81%), ranging from 62% to 100% for specific viruses. In 12 of the 47 patients tested, mVseq identified previously unknown BK virus, human herpesvirus-7, and Epstein–Barr virus infections that were confirmed by qPCR. Conclusions Our results reveal factors that can influence clinical sensitivity, such as high levels of host DNA background and loss of detection in coinfections when 1 virus was at much higher concentration than the others. The mVseq assay is flexible and scalable to incorporate RNA viruses, emerging viruses of interest, and other pathogens important in transplant recipients.

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.

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.

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.

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.

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.

Histologically normal tissue adjacent to the tumor (NAT) is commonly used as a control in cancer studies. However, little is known about the transcriptomic profile of NAT, how it is influenced by the tumor, and how the profile compares with non-tumor-bearing tissues. Here, we integrate data from the Genotype-Tissue Expression project and The Cancer Genome Atlas to comprehensively analyze the transcriptomes of healthy, NAT, and tumor tissues in 6506 samples across eight tissues and corresponding tumor types. Our analysis shows that NAT presents a unique intermediate state between healthy and tumor. Differential gene expression and protein–protein interaction analyses reveal altered pathways shared among NATs across tissue types. We characterize a set of 18 genes that are specifically activated in NATs. By applying pathway and tissue composition analyses, we suggest a pan-cancer mechanism of pro-inflammatory signals from the tumor stimulates an inflammatory response in the adjacent endothelium., Normal tissue adjacent to the tumour (NAT) is often used as a control in cancer studies. Here, the authors analyse across cancer types the transcriptomes of healthy, NAT, and tumour tissues, and find that NAT presents a unique state, potentially due to inflammatory response of the NAT to the tumour tissue.

SummaryType 2 innate lymphoid cells (ILC2s), an innate source of the type 2 cytokines interleukin (IL)-5 and -13, participate in the maintenance of tissue homeostasis. Although type 2 immunity is critically important for mediating metabolic adaptations to environmental cold, the functions of ILC2s in beige or brown fat development are poorly defined. We report here that activation of ILC2s by IL-33 is sufficient to promote the growth of functional beige fat in thermoneutral mice. Mechanistically, ILC2 activation results in the proliferation of bipotential adipocyte precursors (APs) and their subsequent commitment to the beige fat lineage. Loss- and gain-of-function studies reveal that ILC2- and eosinophil-derived type 2 cytokines stimulate signaling via the IL-4Rα in PDGFRα+ APs to promote beige fat biogenesis. Together, our results highlight a critical role for ILC2s and type 2 cytokines in the regulation of adipocyte precursor numbers and fate, and as a consequence, adipose tissue homeostasis.PaperClip

SummaryStore-operated calcium (SOC) channels are vital for activation of the immune cells, and mutations in the channel result in severe combined immunodeficiency in human patients. In lymphocytes, SOC entry is mediated by the Orai1 channel, which is activated by direct binding of STIM1. Here we describe an alternative approach for identifying inhibitors of SOC entry using minimal functional domains of STIM1 and Orai1 to screen a small-molecule microarray. This screen identified AnCoA4, which inhibits SOC entry at submicromolar concentrations and blocks T cell activation in vitro and in vivo. Biophysical studies revealed that AnCoA4 binds to the C terminus of Orai1, directly inhibiting calcium influx through the channel and also reducing binding of STIM1. AnCoA4, unlike other reported SOC inhibitors, is a molecule with a known binding site and mechanism of action. These studies also provide proof of principle for an approach to ion channel drug discovery.

Rapid, accurate, and inexpensive genome sequencing promises to transform medical care. However, a critical hurdle to enabling personalized genomic medicine is predicting the functional impact of novel genomic variation. Various methods of missense variants pathogenicity prediction have been developed by now. Here we present a new strategy for developing a pathogenicity predictor of improved accuracy by applying and training a supervised machine learning model in a gene-specific manner. Our meta-predictor combines outputs of various existing predictors, supplements them with an extended set of stability and structural features of the protein, as well as its physicochemical properties, and adds information about allele frequency from various datasets. We used such a supervised gene-specific meta-predictor approach to train the model on the CFTR gene, and to predict pathogenicity of about 1,000 variants of unknown significance that we collected from various publicly available and internal resources. Our CFTR-specific meta-predictor based on the Random Forest model performs better than other machine learning algorithms that we tested, and also outperforms other available tools, such as CADD, MutPred, SIFT, and PolyPhen-2. Our predicted pathogenicity probability correlates well with clinical measures of Cystic Fibrosis patients and experimental functional measures of mutated CFTR proteins. Training the model on one gene, in contrast to taking a genome wide approach, allows taking into account structural features specific for a particular protein, thus increasing the overall accuracy of the predictor. Collecting data from several separate resources, on the other hand, allows to accumulate allele frequency information, estimated as the most important feature by our approach, for a larger set of variants. Finally, our predictor will be hosted on the ClinGen Consortium database to make it available to CF researchers and to serve as a feasibility pilot study for other Mendelian diseases.

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.

TPS3156 Background: Approximately 7% of advanced solid malignancies have FGFR gene alterations. However, standard treatment for FGFR-altered malignancies has not been established. Moreover, circulating tumor DNA (ctDNA) analysis has a potential to accurately identify FGFR alterations by assessing spatial and temporal intratumoral heterogeneity, which have shown to be associated with a poor prognosis and resistance to anti-cancer therapy. Methods: We are conducting an investigator-initiated multicenter phase II basket-type trial to investigate efficacy and safety of TAS-120, a highly selective covalent pan-FGFR inhibitor, for the patients with advanced solid malignancies with FGFR alterations identified by ctDNA analysis as a part of the Nationwide Cancer Genome Screening Project (GOZILA study, UMIN000029315). Eligibility criteria include histologically confirmed unresectable advanced or recurrent solid tumors regardless of histology of origin; ECOG PS of 0 or 1; refractory or intolerant to the standard therapies; and clonal FGFR alterations ( FGFR1-3 gain-of-function mutations, FGFR1,2 amplifications and FGFR2,3 fusions) identified by a 73-gene sequencing ctDNA panel (Guardant360). Enrolled patients will receive TAS-120 20 mg once daily, orally, in a 21 day-cycle. The primary endpoint is to clarify objective response rate (ORR) assessed by investigators per RECIST v1.1. The secondary endpoints are to evaluate progression-free survival, duration of response, time to treatment failure, disease control rate, overall survival, ORR by central determination, and incidence of adverse events. Target sample size is determined as 26 to test the null hypothesis of ORR as 5% with one-sided alpha level of 2.5% and power of 80% to detect an expected value of ORR as 25%. Furthermore, tumor tissue and ctDNA will be serially collected and analyzed to investigate the resistance mechanisms and provide clinically meaningful biomarker which may be used for identifying and implementing treatment changes. Clinical trial information: 194624.

65 Background: Trastuzumab (T) + chemo is standard first-line therapy (tx) for HER2+ gastroesophageal adenocarcinoma (GEA) pts, though progression ensues in 6-8 months. The approved second-line tx is ramucirumab +/- paclitaxel (R+PAC). Pts with GC are less responsive to R+PAC than gastroesophageal junction (GEJ) pts, in particular HER2+ GC, and no anti-HER2 agents are approved in post-T setting. We report results of combination M+P in HER2+ GC pts and describe a biological rationale for this population. M is an anti-HER2 mAb Fc optimized for enhanced binding to activating FcgRIIIa (CD16A) and decreased binding to inhibitory FcgRIIb (CD32B). M demonstrated an enhanced Fc-dependent MoA, including enhanced ADCC. Methods: HER2+, PD-L1-unselected, second-line GEA pts post T progression received M (15 mg/kg) + P (200 mg) Q3wk. Safety, objective response rate (ORR), median overall & progression-free survival (mOS, mPFS), disease control rate (DCR), circulating tumor DNA, & tumor PD-L1 expression were assessed. Results: To date, 66 GEA pts were dosed; 35 (53%) GC and 31 (47%) GEJ. Overall, 12/66 (18.2%) had tx-related adverse events ≥ grade 3; 5 had drug-related SAEs: dehydration, diabetic ketoacidosis, hypotension and pneumonitis, each a single event, and 2 events of autoimmune hepatitis. Eligibility was based on archival HER2 expression; an exploratory endpoint measured retention of HER2 expression post-T by ERBB2 ctDNA. HER2 expression was lost in 23/56 (41.1%) of pts tested post T. HER2 retention was higher in pts with GC versus GEJ (65.8% vs. 44.8%) and in GEA pts with IHC 3+ vs 2+ archival tumors (61.7% vs 47.4%, respectively). Furthermore, GC had higher PD-L1 expression than GEJ, 53.3 vs. 33.3%, respectively. This coincided with more responses in IHC3+ GC pts, ORR 12/29 (41.4%; 95% CI 23.5-61.1), DCR 21/29 (72.4%; 95% CI 52.8-87.3), mPFS 5.5 months (95% CI 2.3-7.6), mOS not reached, with lower bound of 9.1 months for 95% CI. Enrollment of an additional 25 pts enriched for IHC3+ GC is ongoing. Conclusions: Results suggest that M+P, a chemo-free regimen, demonstrates acceptable tolerability and has encouraging preliminary activity in second-line HER2+ GEA, specifically in GC pts who retain ERBB2 amp prior to second-line tx. Clinical trial information: NCT02689284.

TPS179 Background: Tumor mutational burden (TMB) is an emerging biomarker for immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC). Analysis of circulating tumor DNA (ctDNA) has been reported to effectively identify patients likely to respond to ICIs by effectively and non-invasively evaluating the TMB in the tumor in NSCLC and gastric cancer. Methods: We are conducting an investigator-initiated multicenter phase II basket trial to investigate efficacy and safety of nivolumab monotherapy in patients with advanced gastrointestinal (GI) cancers with high bTMB identified by ctDNA analysis as part of the Nationwide Cancer Genome Screening Project (SCRUM-Japan GI-SCREEN). Eligibility criteria include histologically confirmed unresectable or recurrent GI malignancies; ECOG PS of 0 or 1; refractory or intolerant to the standard therapies; and high bTMB identified by a 73-gene sequencing ctDNA panel (Guardant360) regardless of microsatellite instability status. Patients will be enrolled into one of four disease-specific cohorts (colorectal, gastric, esophageal, and other GI cancer cohort), and receive intravenous nivolumab monotherapy of 360 mg every 3 weeks. The bTMB score is calculated by adjustment of mutation count by tumor fraction, and tentative bTMB level cut-offs were determined according to objective response rate (ORR) reported for ICI treatment for each tumor subtype in previous trials. The trial will utilize a two-stage design with a Bayesian hierarchical model, and tentative bTMB level cut-off will be re-assessed in the first stage. Primary endpoint in each stage is the disease control rate at 6 week and the ORR assessed by investigators per RECIST v1.1, respectively. Target sample size is determined as 70 in total so that the statistical power in each disease-specific cohort calculated based on a Bayesian posterior distribution attains 70 to 80% with one-sided alpha level in each cohort of approximately 10%. For biomarker analysis, tumor tissue and ctDNA will be serially collected and analyzed by whole-exome, transcriptome, and T cell receptor sequencing. This trial was initiated since September 2018. Clinical trial information: UMIN000033182.

For placental mammals, the transition from the in utero maternal environment to postnatal life requires the activation of thermogenesis to maintain their core temperature. This is primarily accomplished by induction of uncoupling protein 1 (UCP1) in brown and beige adipocytes, the principal sites for uncoupled respiration. Despite its importance, how placental mammals license their thermogenic adipocytes to participate in postnatal uncoupled respiration is not known. Here, we provide evidence that the "alarmin" IL-33, a nuclear cytokine that activates type 2 immune responses, licenses brown and beige adipocytes for uncoupled respiration. We find that, in absence of IL-33 or ST2, beige and brown adipocytes develop normally but fail to express an appropriately spliced form of Ucp1 mRNA, resulting in absence of UCP1 protein and impairment in uncoupled respiration and thermoregulation. Together, these data suggest that IL-33 and ST2 function as a developmental switch to license thermogenesis during the perinatal period. PAPERCLIP.

Resistin-like molecules (RELMs) are highly expressed following helminth infection, where they impact both the host and helminth. While RELMα ( Retnla ) impairs helminth expulsion by inhibiting protective Th2 immunity, RELMβ ( Retnlb ) can promote its expulsion. We employed Retnla −/− and Retnlb −/− mice to delineate the function of both proteins following infection with Nippostrongylus brasiliensis , a hookworm that infects the lung and intestine. Whereas wild-type (WT) and Retnlb −/− mice exhibited equivalent infection-induced inflammation, Retnla −/− mice suffered a heightened inflammatory response, including increased mortality, weight loss, and lung inflammation. In the intestine, Retnla −/− mice had low parasite egg burdens compared to those of WT mice, while Retnlb −/− mice exhibited high egg burdens, suggesting that RELMα and RELMβ have functionally distinct effects on immunity and inflammation to N. brasiliensis . To test the importance of both proteins, we generated Retnla −/− Retnlb −/− mice. Infected Retnla −/− Retnlb −/− mice exhibited similar responses to Retnla −/− mice, including increased mortality and lung inflammation. This inflammatory response in Retnla −/− Retnlb −/− mice negatively impacted N. brasiliensis fitness, as demonstrated by significantly lower worm ATP levels and decreased intestinal worm burden and fecundity. Lung cytokine analysis revealed that Retnla −/− and Retnla −/− Retnlb −/− mice expressed significantly increased levels of interleukin-4 (IL-4). Finally, we generated Retnla −/− mice on the Rag −/− background and observed that the effects of RELMα were abrogated in the absence of adaptive immunity. Together, these data demonstrate that RELMα but not RELMβ significantly impacts the immune response to N. brasiliensis infection by downregulating the Th2 adaptive immune response in the lung, which protects the host but allows improved parasite fitness.

Analyses of whole organs from parasite-infected animals can reveal the entirety of the host tissue transcriptome, but conventional approaches make it difficult to dissect out the contributions of individual cellular subsets to observed gene expression. Computational deconvolution of gene expression data may be one solution to this problem. We tested this potential solution by deconvoluting whole bladder gene expression microarray data derived from a model of experimental urogenital schistosomiasis. A supervised technique was used to group B-cell and T-cell related genes based on their cell types, with a semi-supervised technique to calculate the proportions of urothelial cells. We demonstrate that the deconvolution technique was able to group genes into their correct cell types with good accuracy. A clustering-based methodology was also used to improve prediction. However, incorrectly predicted genes could not be discriminated using this methodology. The incorrect predictions were primarily IgH- and IgK-related genes. To our knowledge, this is the first application of computational deconvolution to complex, parasite-infected whole tissues. Other computational techniques such as neural networks may need to be used to improve prediction.

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...

Complex interplay between T helper (Th) cells and macrophages contributes to the formation and progression of atherosclerotic plaques. While Th1 cytokines promote inflammatory activation of lesion macrophages, Th2 cytokines attenuate macrophage-mediated inflammation and enhance their repair functions. In spite of its biologic importance, the biochemical and molecular basis of how Th2 cytokines promote maturation of anti-inflammatory macrophages is not understood. We show here that in response to interleukin-4 (IL-4), signal transducer and activator of transcription 6 (STAT6) and PPARgamma-coactivator-1beta (PGC-1beta) induce macrophage programs for fatty acid oxidation and mitochondrial biogenesis. Transgenic expression of PGC-1beta primes macrophages for alternative activation and strongly inhibits proinflammatory cytokine production, whereas inhibition of oxidative metabolism or RNAi-mediated knockdown of PGC-1beta attenuates this immune response. These data elucidate a molecular pathway that directly links mitochondrial oxidative metabolism to the anti-inflammatory program of macrophage activation, suggesting a potential role for metabolic therapies in treating atherogenic inflammation.

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]

SummaryBeige fat, which expresses the thermogenic protein UCP1, provides a defense against cold and obesity. Although a cold environment is the physiologic stimulus for inducing beige fat in mice and humans, the events that lead from the sensing of cold to the development of beige fat remain poorly understood. Here, we identify the efferent beige fat thermogenic circuit, consisting of eosinophils, type 2 cytokines interleukin (IL)-4/13, and alternatively activated macrophages. Genetic loss of eosinophils or IL-4/13 signaling impairs cold-induced biogenesis of beige fat. Mechanistically, macrophages recruited to cold-stressed subcutaneous white adipose tissue (scWAT) undergo alternative activation to induce tyrosine hydroxylase expression and catecholamine production, factors required for browning of scWAT. Conversely, administration of IL-4 to thermoneutral mice increases beige fat mass and thermogenic capacity to ameliorate pre-established obesity. Together, our findings have uncovered the efferent circuit controlling biogenesis of beige fat and provide support for its targeting to treat obesity.

Vertebrate tissues comprise precise admixtures of parenchymal and hematopoietic cells, whose interactions are vital to proper tissue function. By regulating this interaction, vertebrates are able to mitigate environmental stress and coordinate dramatic physiologic adaptations. For instance, under conditions of chronic nutrient excess, leukocyte recruitment and activation increase in an effort to decrease excess nutrient storage and alleviate adipocyte stress. While basal equilibria may be reestablished upon normalization of nutrient intake, a new set point characterized by insulin resistance and chronic inflammation is established if the stress persists. Consequently, although this response is adaptive in settings of acute overfeeding and infection, it has catastrophic health consequences in the modern context of obesity. Understanding how leukocyte set points (numbers and activation status) are established, maintained, and regulated in tissues is, thus, critical to our understanding of, and intervention in, chronic metabolic diseases, such as obesity and diabetes.

Urogenital schistosomiasis, chronic infection by Schistosoma haematobium, affects 112 million people worldwide. S. haematobium worm oviposition in the bladder wall leads to granulomatous inflammation, fibrosis, and egg expulsion into the urine. Despite the global impact of urogenital schistosomiasis, basic understanding of the associated pathologic mechanisms has been incomplete due to the lack of suitable animal models. We leveraged our recently developed mouse model of urogenital schistosomiasis to perform the first-ever profiling of the early molecular events that occur in the bladder in response to the introduction of S. haematobium eggs. Microarray analysis of bladders revealed rapid, differential transcription of large numbers of genes, peaking three weeks post-egg administration. Many differentially transcribed genes were related to the canonical Type 2 anti-schistosomal immune response, as reflected by the development of egg-based bladder granulomata. Numerous collagen and metalloproteinase genes were differentially transcribed over time, revealing complex remodeling and fibrosis of the bladder that was confirmed by Masson's Trichrome staining. Multiple genes implicated in carcinogenesis pathways, including vascular endothelial growth factor-, oncogene-, and mammary tumor-related genes, were differentially transcribed in egg-injected bladders. Surprisingly, junctional adhesion molecule, claudin and uroplakin genes, key components for maintaining the urothelial barrier, were globally suppressed after bladder exposure to eggs. This occurred in the setting of urothelial hyperplasia and egg shedding in urine. Thus, S. haematobium egg expulsion is associated with intricate modulation of the urothelial barrier on the cellular and molecular level. Taken together, our findings have important implications for understanding host-parasite interactions and carcinogenesis in urogenital schistosomiasis, and may provide clues for novel therapeutic strategies., Author Summary Parasitic Schistosoma haematobium worms cause urogenital schistosomiasis in 112 million people worldwide. These worms lay eggs in the bladder wall, resulting in inflammation, fibrosis (internal scarring), bladder cancer, and passage of eggs into the urine. Indeed, the International Agency for Research on Cancer within the World Health Organization has classified S. haematobium as a “Class I” agent (“Carcinogenic to humans”). Moreover, S. haematobium-induced fibrosis and resulting obstructive kidney failure leads to 150,000 deaths annually. As a result, S. haematobium infection is one of the most important causes of worm-related death globally. In spite of this, research on this parasite is sparse due to a lack of suitable animal models. We have used our recently developed mouse model of urogenital schistosomiasis to understand the global bladder gene response to this infection. Large numbers of genes featured differential transcription after experimental infection, including specific immune response-, fibrosis-, cancer-, and bladder function-related genes. The relevance of these gene-based findings was verified through microscopic examination of egg-exposed bladders. Our data will improve our comprehension of urogenital schistosomiasis, and may help identify new targets for diagnosis and treatment of this disease, and possibly bladder cancer and bladder-based inflammatory disorders as well.

Macrophages rapidly engulf apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune responses against self antigens. Although factors participating in recognition and engulfment of apoptotic cells have been identified, the transcriptional basis for the sensing and the silent disposal of apoptotic cells is unknown. Here we show that peroxisome proliferator-activated receptor-delta (PPAR-delta) is induced when macrophages engulf apoptotic cells and functions as a transcriptional sensor of dying cells. Genetic deletion of PPAR-delta decreases expression of opsonins such as complement component-1qb (C1qb), resulting in impairment of apoptotic cell clearance and reduction in anti-inflammatory cytokine production. This increases autoantibody production and predisposes global and macrophage-specific Ppard(-/-) mice to autoimmune kidney disease, a phenotype resembling the human disease systemic lupus erythematosus. Thus, PPAR-delta has a pivotal role in orchestrating the timely disposal of apoptotic cells by macrophages, ensuring that tolerance to self is maintained.

Schistosoma haematobium is the etiologic agent for urogenital schistosomiasis, a major source of morbidity and mortality for more than 112 million people worldwide. Infection with S. haematobium results in a variety of immunopathologic sequelae caused by parasite oviposition within the urinary tract, which drives inflammation, hematuria, fibrosis, bladder dysfunction, and increased susceptibility to urothelial carcinoma. While humans readily develop urogenital schistosomiasis, the lack of an experimentally-tractable model has greatly impaired our understanding of the mechanisms that underlie this important disease. We have developed an improved mouse model of S. haematobium urinary tract infection that recapitulates several aspects of human urogenital schistosomiasis. Following microinjection of purified S. haematobium eggs into the bladder wall, mice consistently develop macrophage-rich granulomata that persist for at least 3 months and pass eggs in their urine. Importantly, egg-injected mice also develop urinary tract fibrosis, bladder dysfunction, and various urothelial changes morphologically reminiscent of human urogenital schistosomiasis. As expected, S. haematobium egg-induced immune responses in the immediate microenvironment, draining lymph nodes, and systemic circulation are associated with a Type 2-dominant inflammatory response, characterized by high levels of interleukin-4, eosinophils, and IgE. Taken together, our novel mouse model may help facilitate a better understanding of the unique pathophysiological mechanisms of epithelial dysfunction, tissue fibrosis, and oncogenesis associated with urogenital schistosomiasis., Author Summary Urogenital schistosomiasis (infection with parasitic Schistosoma haematobium worms, the most common human-specific Schistosoma species globally) affects over 112 million people worldwide. S. haematobium worms primarily lay eggs in the bladder, upper urinary and genital tracts, and the host immune response to these eggs is considered to cause almost all associated disease in these organs. Resulting conditions include hematuria (bloody urine), urinary frequency, fibrosis (internal scarring) of the urinary tract, increased risk of bladder cancer, and enhanced susceptibility to contracting HIV. Approximately 150,000 people die annually from S. haematobium-induced obstructive kidney failure alone, making this species one of the deadliest worms worldwide. Despite the importance of S. haematobium, a lack of an experimentally manipulable model has contributed to the paucity of research focusing on this parasite. We have circumvented the barriers to natural S. haematobium oviposition in the mouse bladder by directly microinjecting parasite eggs into the bladder wall. This triggers inflammation, hematuria, urinary frequency, fibrosis, egg shedding, and epithelial changes that are similar to that seen in clinical S. haematobium infections. Our model may provide new opportunities to better understand the basic molecular and cellular immunology of urogenital schistosomiasis and thereby contribute to the development of new diagnostics and therapeutics.

Mammals possess a remarkable ability to maintain and defend a constant internal milieu against diverse environmental threats. Unsurprisingly, the two systems tasked with these duties, metabolism and immunity, have evolved to share a common modular architecture that allows extensive bidirectional communication and coordination. Indeed, recent observations have highlighted numerous, functionally critical immune regulatory modules located within diverse metabolic circuits. In this Review, we discuss the architectural commonality between immunity and metabolism, and highlight how these two primordially disparate systems leverage shared regulatory axes to coordinate metabolic physiology under conditions of normality and chronic overnutrition. Such an integrated perspective both advances our understanding of basic physiology and highlights potential opportunities for therapeutic intervention in metabolic dysfunction.

Newborn screening for cystic fibrosis enables early detection and management of this debilitating genetic disease. Implementing comprehensive CFTR analysis using Sanger sequencing as a component of confirmatory testing of all screen-positive newborns has remained impractical due to relatively lengthy turnaround times and high cost. Here, we describe CFseq, a highly sensitive, specific, rapid (2000 CFTR variants. Validation studies across 190 dried blood spots demonstrated 100% sensitivity and a positive predictive value of 100% for single-nucleotide variants and insertions and deletions and complete concordance across the polymorphic poly-TG and consecutive poly-T tracts. Additionally, we accurately detected both a known exon 2,3 deletion and a previously undetected exon 22,23 deletion. CFseq is thus able to replace all existing CFTR molecular assays with a single robust, definitive assay at significant cost and time savings and could be adapted to high-throughput screening of other inherited conditions.

Chemotherapy-induced hemorrhagic cystitis is characterized by bladder pain and voiding dysfunction caused by hemorrhage and inflammation. Novel therapeutic options to treat hemorrhagic cystitis are needed. We previously reported that systemic administration of the Schistosomiasis hematobium-derived protein H-IPSEH06 (IL-4-inducing principle from Schistosoma mansoni eggs) is superior to three doses of MESNA in alleviating hemorrhagic cystitis (Mbanefo EC, Le L, Pennington LF, Odegaard JI, Jardetzky TS, Alouffi A, Falcone FH, Hsieh MH. FASEB J 32: 4408–4419, 2018). Based on prior reports by others on S. mansoni IPSE (M-IPSE) and additional work by our group, we reasoned that H-IPSE 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. hematobium IPSE ortholog IPSEH03, hereafter called H-IPSEH03, might be more efficacious in preventing hemorrhagic cystitis compared with systemic administration of IPSEH06. We report that H-IPSEH03, like M-IPSE and H-IPSEH06, activates IgE-bearing basophils in a nuclear factor of activated T-cells reporter assay, indicating activation of the cytokine pathway. Furthermore, H-IPSEH03 attenuates ifosfamide-induced increases in bladder wet weight in an IL-4-dependent fashion. H-IPSEH03 relieves hemorrhagic cystitis-associated allodynia and modulates voiding patterns in mice. Finally, H-IPSEH03 drives increased urothelial cell proliferation, suggesting that IPSE induces bladder repair mechanisms. Taken together, H-IPSEH03 may be a potential novel therapeutic to treat hemorrhagic cystitis by basophil activation, attenuation of allodynia, and promotion of urothelial cell proliferation.