Citations

Abstract CCGA [NCT02889978] is the largest study of cfDNA-based early cancer detection; the first CCGA learnings from multiple cfDNA assays are reported here. This prospective, multi-center, observational study has enrolled 10,012 of 15,000 demographically-balanced participants at 141 sites. Blood was collected from participants with newly diagnosed therapy-naive cancer (C, case) and participants without a diagnosis of cancer (noncancer [NC], control) as defined at enrollment. This preplanned substudy included 878 cases, 580 controls, and 169 assay controls (n=1627) across 20 tumor types and all clinical stages. All samples were analyzed by: 1) Paired cfDNA and white blood cell (WBC)-targeted sequencing (60,000X, 507 gene panel); a joint caller removed WBC-derived somatic variants and residual technical noise; 2) Paired cfDNA and WBC whole-genome sequencing (WGS; 35X); a novel machine learning algorithm generated cancer-related signal scores; joint analysis identified shared events; and 3) cfDNA whole-genome bisulfite sequencing (WGBS; 34X); normalized scores were generated using abnormally methylated fragments. In the targeted assay, non-tumor WBC-matched cfDNA somatic variants (SNVs/indels) accounted for 76% of all variants in NC and 65% in C. Consistent with somatic mosaicism (i.e., clonal hematopoiesis), WBC-matched variants increased with age; several were non-canonical loss-of-function mutations not previously reported. After WBC variant removal, canonical driver somatic variants were highly specific to C (e.g., in EGFR and PIK3CA, 0 NC had variants vs 11 and 30, respectively, of C). Similarly, of 8 NC with somatic copy number alterations (SCNAs) detected with WGS, 4 were derived from WBCs. WGBS data revealed informative hyper- and hypo-fragment level CpGs (1:2 ratio); a subset was used to calculate methylation scores. A consistent “cancer-like” signal was observed in <1% of NC participants across all assays (representing potential undiagnosed cancers). An increasing trend was observed in NC vs stages I-III vs stage IV (nonsyn. SNVs/indels per Mb [Mean±SD] NC: 1.01±0.86, stages I-III: 2.43±3.98; stage IV: 6.45±6.79; WGS score NC: 0.00±0.08, I-III: 0.27±0.98; IV: 1.95± 2.33; methylation score NC: 0±0.50; I-III: 1.02±1.77; IV: 3.94±1.70). These data demonstrate the feasibility of achieving >99% specificity for invasive cancer, and support the promise of cfDNA assay for early cancer detection. Additional data will be presented on detected plasma:tissue variant concordance and on multi-assay modeling. Citation Format: Alexander A. Aravanis, Geoffrey R. Oxnard, Tara Maddala, Earl Hubbell, Oliver Venn, Arash Jamshidi, Ling Shen, Hamed Amini, John A. Beausang, Craig Betts, Daniel Civello, Konstantin Davydov, Saniya Fazullina, Darya Filippova, Sante Gnerre, Samuel Gross, Chenlu Hou, Roger Jiang, Byoungsok Jung, Kathryn Kurtzman, Collin Melton, Shivani Nautiyal, Jonathan Newman, Joshua Newman, Cosmos Nicolaou, Richard Rava, Onur Sakarya, Ravi Vijaya Satya, Seyedmehdi Shojaee, Kristan Steffen, Anton Valouev, Hui Xu, Jeanne Yue, Nan Zhang, Jose Baselga, Rosanna Lapham, Daron G. Davis, David Smith, Donald Richards, Michael V. Seiden, Charles Swanton, Timothy J. Yeatman, Robert Tibshirani, Christina Curtis, Sylvia K. Plevritis, Richard Williams, Eric Klein, Anne-Renee Hartman, Minetta C. Liu. Development of plasma cell-free DNA (cfDNA) assays for early cancer detection: first insights from the Circulating Cell-Free Genome Atlas Study (CCGA) [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 LB-343.

Abstract Biodistribution detection methods currently hold a central role in evaluating clinical potential of new drugs and nanomedicine in vivo, either through therapeutic efficacy or toxicity assays. However, the explorative nature of these methods makes it practically difficult to validate them for clinical studies to comply with regulations as determined by the Food and Drug Administration. This work proposes a new systematic comparison of biodistribution methods to facilitate clinical validation and employment in pre-clinical and early stage clinical studies. Several optical methods (detection through IVIS, confocal microscopy, fluorescence emission), direct detection methods (detection through ICP-OES), and radiation-based methods (detection through gamma counter) are systematically tested and validated on cGMP grade microparticles. The classification here proposed quantitatively highlights the advantages and disadvantages of each method, expressing them in terms of: limit of detection, recovery rate, reproducibility, and sensitivity. As a result, this quantitative evaluation of several preclinical biodistribution detection methods leads to a new rational and objective classification of biodistribution methods based on quantitative characteristics, which is essential to facilitate clinical translation of pre-clinical data collection. Their different characteristics and application-specific advantages are also discussed based in light of the results presented. Overall, this work proposes a new and rational quantitative comparison of biodistribution detection methods with the goal to provide a guide to facilitate clinical translation of on-going and future pre-clinical and early-stage clinical studies. Citation Format: Sara Nizzero, Feng Li, Alessandro Venuta, Guodong Zhang, Carlotta Borsoi, Joy Wolfram, Junhua Mai, Haifa Shen, Zheng Li, Elvin Blanco, Mauro Ferrari. Empowering preclinical studies: A systematic and quantitative analysis of biodistribution methods to facilitate clinical translation of new drugs [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 LB-019.

Clearance of protein-bound uremic toxins (PBUTs) by dialysis is a challenge in the treatment of uremic patients. Shen-Shuai-Ning (SSN), a traditional Chinese medicine formulation, has been used commonly in China to retard kidney disease progression and decrease uremic toxins in chronic kidney disease (CKD) patients, but the effects of SSN on serum PBUTs in dialysis patients were not investigated. We conducted a randomized controlled trial in patients on peritoneal dialysis (PD) at dialysis center of Changzheng Hospital to evaluate the effects of SSN on serum PBUTs. Participants with SSN intervention took 5 g SSN granule three times daily for 12 weeks, while the baseline medications and dialysis prescriptions remained during the study in all patients. The serum concentrations of indoxyl sulphate (IS) and p-cresol sulphate (PCS) were determined by HPLC/MS/MS and biochemical parameters were assessed during the study. Sixty PD patients were enrolled and randomly allocated into SSN group and control group. Total IS level was significantly lower in SSN group than in control group at week 4, 8, and 12 (27.28 ± 18.19, 29.73 ± 19.10, and 29.41 ± 17.61 mg/l compared with 39.25 ± 20.23, 44.86 ± 23.91, and 45.34 ± 33.52 mg/l, respectively). However, there were no statistical difference of total PCS, free forms of IS and PCS concentrations between SSN group and control group during 12 weeks follow-up. Administration of SSN granule orally decreased serum total IS level effectively in uremic patients on PD with good tolerance. Benefits of PD patients’ outcomes from IS reduction by SSN awaits further large size and long duration clinical trials to verify.

Abstract Researchers hypothesized that telomere shortening facilitates carcinogenesis. Previous studies found inconsistent associations between blood leukocyte telomere length (LTL) and cancer. Epigenetic reprogramming of telomere maintenance mechanisms may help explain this inconsistency. We examined associations between DNA methylation in telomere-related genes (TRG) and cancer. We analyzed 475 participants providing 889 samples 1 to 3 times (median follow-up, 10.1 years) from 1999 to 2013 in the Normative Aging Study. All participants were cancer-free at each visit and blood leukocytes profiled using the Illumina 450K array. Of 121 participants who developed cancer, 34 had prostate cancer, 10 melanoma, 34 unknown skin malignancies, and 43 another cancer. We examined 2,651 CpGs from 80 TRGs and applied a combination of Cox and mixed models to identify CpGs prospectively associated with cancer (at FDR < 0.05). We also explored trajectories of DNA methylation, logistic regression stratified by time to diagnosis/censoring, and cross-sectional models of LTL at first blood draw. We identified 30 CpGs on 23 TRGs whose methylation was positively associated with cancer incidence (β = 1.0–6.93) and one protective CpG in MAD1L1 (β = −0.65), of which 87% were located in TRG promoters. Methylation trajectories of 21 CpGs increased in cancer cases relative to controls; at 4 to 8 years prediagnosis/censoring, 17 CpGs were positively associated with cancer. Three CpGs were cross-sectionally associated with LTL. TRG methylation may be a mechanism through which LTL dynamics reflect cancer risk. Future research should confirm these findings and explore potential mechanisms underlying these findings, including telomere maintenance and DNA repair dysfunction. Cancer Prev Res; 11(8); 511–22. ©2018 AACR.

LBA8501 Background: Plasma cfDNA genomic analysis is used widely for the care of advanced lung cancer, but its suitability for early stage lung cancer detection is not well established. CCGA (NCT02889978) is a prospective, multi-center, observational study launched for the development of a noninvasive assay for cancer detection. Methods: Blood was prospectively collected (N = 1627) from 749 controls (no cancer diagnosis) and 878 participants (pts) with newly-diagnosed untreated cancer in this preplanned substudy, including 127 pts with lung cancer. Three prototype sequencing assays were performed: paired cfDNA and white blood cell (WBC) targeted sequencing (507 genes, 60,000X) for single nucleotide variants/indels; paired cfDNA and WBC whole genome sequencing (WGS) for copy number variation (30X); and cfDNA whole genome bisulfite sequencing (WGBS) for methylation (30X). For each assay, a classification model using 10-fold cross-validation was developed for all pts with cancer, then evaluated in the pts with lung cancer; sensitivity was estimated at 95% specificity. Results: We evaluated pts with lung cancer (127) and a subset of controls (580) with similar ages (mean±SD yrs: 67±9, 60±13), 85% and 43% were ever-smokers, and 46% and 22% were men, respectively. Of 3055 nonsynonymous mutations detected across 122 evaluable pts with lung cancer, > 50% were detected in WBC consistent with clonal hematopoiesis (CH). Accounting for CH, sensitivity in 63 stage I-IIIA pts evaluable across all 3 assays was 48% (35-61, targeted), 54% (41-67, WGS), and 56% (43-68, WGBS); in 54 stage IIIB-IV pts it was 85% (73-93, targeted), 91% (80-97, WGS), and 93% (82-98, WGBS) . Similar sensitivities were observed across histological subtypes (adenocarcinoma, squamous cell, small cell). Comparison to tumor WGS and multi-assay classification will be reported. Conclusions: Early stage lung cancers are detectable in cfDNA using a genome-wide sequencing approach. For lung cancer detection using targeted assays, CH must be accounted for to minimize false positives. Assay optimization is ongoing to allow further clinical development in the intended use population. Clinical trial information: NCT02889978.

Abstract Background We quantified loss of response (LOR) to vedolizumab (VDZ) in clinical practice and assessed the effectiveness of VDZ dose intensification for managing LOR. Methods Retrospective review (May 2014–December 2016) of a prospectively maintained inflammatory bowel disease (IBD) registry. Kaplan-Meier estimates were used to determine rates of LOR to VDZ . Independent predictors of LOR were identified using univariate and multivariable Cox proportional hazard regression. Success of recapturing response (>50% reduction in symptoms from baseline) and remission (complete resolution of symptoms) after dose intensification was quantified. Results Cumulative rates for VDZ LOR were 20% at 6 months and 35% at 12 months, with slightly lower rates in Crohn’s disease than in ulcerative colitis (6 months 15% vs 18% and 12 months 30% vs 39%, P = 0.03). On multivariable analysis, LOR to a tumor necrosis factor (TNF) antagonist before VDZ use was associated with an increased risk for LOR to VDZ [hazard ratio (HR) 1.93; 95% confidence interval (CI) 1.25–2.97] in all patients. For Crohn’s disease patients specifically, higher baseline C-reactive protein concentration was associated with increased risk for LOR to VDZ (HR 1.01 per mg/dL increase, 95% CI 1.01–1.02). Shortening of VDZ infusion interval from 8 to every 4 or 6 weeks recaptured response in 49% and remission in 18% of patients. Conclusions LOR to a TNF antagonist before VDZ use and higher baseline C-reactive protein are important predictors of VDZ LOR. Treatment response can be recaptured in almost half of these patients with VDZ infusion interval shortening.

PurposeThe purpose of this study was to develop a fast method for proton range quality assurance (QA) using a dual step wedge and two‐dimensional (2D) scintillator and to evaluate the robustness, sensitivity, and long‐term reproducibility of this method.MethodsAn in‐house customized dual step wedge and a 2D scintillator were developed to measure proton ranges. Proton beams with homogenous fluence were delivered through wedge, and the images captured by the scintillator were used to calculate the proton ranges by a simple trigonometric method. The range measurements of 97 energies, comprising all clinically available synchrotron energies at our facility (ranges varying from 4 to 32 cm) were repeated ten times in all four gantry rooms for range baseline values. They were then used for evaluating room‐to‐room range consistencies. The robustness to setup uncertainty was evaluated by measuring ranges with ±2 mm setup deviations in the x, y, and z directions. The long‐term reproducibility was evaluated by 1 month of daily range measurements by this method.ResultsRanges of all 97 energies were measured in less than 10 minutes including setup time. The reproducibility in a single day and daily over 1 month is within 0.1 and 0.15 mm, respectively. The method was very robust to setup uncertainty, with measured range consistencies within 0.15 mm for ±2 mm couch shifts. The method was also sensitive enough for validating range consistencies among gantry rooms and for detecting small range variations.ConclusionsThe new method of using a dual step wedge and scintillator for proton range QA was efficient, highly reproducible, and robust. This method of proton range QA was highly feasible and appealing from a workflow point of view.

ABSTRACT Osteoclasts are bone‐resorbing cells differentiated from macrophage/monocyte precursors in response to macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL). In vitro models are principally based on primary bone marrow macrophages (BMMs), but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior. In particular, M‐CSF is a prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form osteoclasts in the presence of RANKL, but M‐CSF is not required. Based on these key differences, we sought to understand their functional implications and how it might affect osteoclast differentiation and related signaling pathways. Using a robust and high‐throughput proteomics strategy, we quantified the global protein changes in osteoclasts derived from BMMs and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Data are available via ProteomeXchange with the identifier PXD009610. Correlation analysis of the proteomes demonstrated low concordance between the two cell types ( R 2 ≈ 0.13). Bioinformatics analysis indicate that RANKL‐dependent signaling was intact in RAW 264.7 cells, but biological processes known to be dependent on M‐CSF were significantly different, including cell cycle control, cytoskeletal organization, and apoptosis. RAW 264.7 cells exhibited constitutive activation of Erk and Akt that was dependent on the activity of Abelson tyrosine kinase, and the timing of Erk and Akt activation was significantly different between BMMs and RAW 264.7 cells. Our findings provide the first evidence for major discrepancies between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line for studying M‐CSF‐dependent signaling and functions. © 2018 American Society for Bone and Mineral Research. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.