Serial Blood Collection Research Articles

Abstract 5534: Analysis of TERT mutant circulating tumor DNA as a potential biomarker of disease activity in patients with unresectable stage III/IV melanoma receiving immuno-oncology therapies

Abstract Purpose: Our goal is to develop response predictive biomarkers for melanoma patients that are more sensitive than standard measures of disease activity. TERT promoter mutations occur frequently in many cancers, especially melanoma. We developed droplet digital PCR assays that detect the 2 most frequently occurring TERT promoter mutations, C228T and C250T. We previously demonstrated that these assays could detect TERTmutant cell-free, circulating tumor DNA (ctDNA) in the plasma of metastatic melanoma patients. In the current study we sought to compare the clinical sensitivity and specificity of these blood-based assays to the current blood biomarker, lactate dehydrogenase (LDH), in detecting the presence of metastatic melanoma and disease progression among patients with unresectable Stage III/IV disease. Methods: We analyzed 71 patients' tumors with unresectable stage III/IV metastatic melanoma undergoing treatment with immuno-oncology therapies, prospectively acquired by the NYU Interdisciplinary Melanoma Cooperative Group. TERT mutations were identified in tumors using droplet digital PCR (ddPCR) or SNaPShot assays (prior to the development of the ddPCR assays). Patients with TERT mutant tumors were selected for further study based on the availability of serial blood collections and radiographic scan data. ddPCR assays used to analyze ctDNA from these patients were chosen based on the tumor mutation detected. Plasmas used for ctDNA analysis ranged from 1-5 mL (73% having at least 4 mL). The ctDNA results were compared with LDH levels and disease status based on radiographic scans and clinical notes. Results: We identified TERT mutations in 31/71 (44%) tumors (18/31 C228T and 13/31 C250T). Our final data set consisted of 26 patients with 85 plasmas. Among patients with available pretreatment samples, ctDNA and LDH were elevated in 14/24 (58%) and 5/24 (21%) patients, respectively (p-value=0.012, McNemar exact test). Among patients whose disease progressed, ctDNA and LDH were elevated at the time of progression in 11/16 (69%) and 4/16 (25%) patients, respectively (p=0.039). Among the patients who had either a complete response (n=2), partial response (n=3), or stable disease (n=3), 1/8 had a slightly elevated ctDNA (3.8 copies/mL with normal level <3 copies/mL). None of the patients had an elevated LDH. One patient had a mixed response associated with an elevated ctDNA (13.2 copies/mL) and RECIST score equal to 3.4 cm, and then the ctDNA dropped below baseline when the RECIST measurement fell to 2.8 cm on a follow-up scan. Conclusion: These data demonstrate that ddPCR assays detecting TERT C228T or C250T mutations in ctDNA of metastatic melanoma patients have a greater sensitivity to detect the presence of metastatic disease and disease progression than the currently used standard, LDH. Citation Format: Mahrukh M. Syeda, Broderick Corless, Melissa Wilson, Yesung Lee, Jeremy Tchack, Todd Wechter, Una Moran, George Karlin-Neumann, Anna C. Pavlick, Iman Osman, Yongzhao Shao, David Polsky. Analysis of TERTmutant circulating tumor DNA as a potential biomarker of disease activity in patients with unresectable stage III/IV melanoma receiving immuno-oncology therapies [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 5534.

EGFR Amplification as a Target in Gastroesophageal Adenocarcinoma: Do Anti-EGFR Therapies Deserve a Second Chance?

<b/> Anti-EGFR therapies have failed to improve survival for unselected patients with metastatic gastroesophageal cancer, but in a subset of patients, EGFR amplification may predict treatment benefit. Maron and colleagues report the clinical activity of anti-EGFR therapies in a cohort of patients with EGFR-amplified metastatic gastroesophageal cancer and utilize serial blood and tumor tissue collection to identify molecular drivers of treatment sensitivity and resistance. Their insights offer a path to overcome technical limitations associated with EGFR amplification and facilitate molecularly targeted therapeutic strategies. Cancer Discov; 8(6); 679-81. ©2018 AACRSee related article by Maron et al., p. 696.

Pharmacokinetic profile and oral bioavailability of Kaurenoic acid from Copaifera spp. in rats

Kaurenoic acid (KA) is a kaurane diterpene found in several medicinal plants that displays biological activities, such as anti-inflammatory, smooth muscle relaxant and hypotensive response. However, there are no pharmacokinetic data available about this molecule. The purpose of the study was to determine the pharmacokinetic profile and the oral bioavailability of KA in rats. Wistar rats submitted to jugular vein cannulation received 50 mg/kg of KA by intravenous or oral route. The implanted cannula allowed intravenous administration and serial blood collection along 10 h. Analytical quantification was performed by reversed phase HPLC-UV and mobile phase composed by acetonitrile:acidified water (70:30 v/v). The validated analytical method showed precision, accuracy, robustness, reliability and linearity between 0.75 and 100 μg/mL. KA administered intravenously showed a linear and two-compartment kinetic behavior at the tested dose. The following pharmacokinetic parameters were determined: Cmax = 22.17 ± 1.65 mg/L; V = 14.53 ± 1.47 L/kg; CL = 17.67 ± 1.50 mL/min/kg; AUC0-∞ = 2859.65 ± 278.42 mg·min/L, K = 0.073 ± 0.005 h−1 and t1/2β = 9.52 ± 0.61 h. Oral treatment did not provide detectable plasma levels of KA, avoiding the determination of its bioavailability.

Circulating estrogen receptor (ER) mutations and splice variants in men with advanced prostate cancer (APC).

363 Background: Circulating DNA and RNA have been studied in APC as biomarkers of potential clinical significance. Candidates include androgen receptor amplification or emergence of splice variants. ER signalling has been associated with prostate cancer progression in vitro but blood-based detection of ER variants has not been studied. Methods: Men with APC were recruited for serial blood collection during routine management, from which cell-free DNA (cfDNA) and cell-free RNA (cfRNA) were extracted. Six well-described ERα mutations (E380Q, L536Q, Y537C, Y537S, Y537N &amp; D538G) and six ER splice variants (ERα-66, ERα-36, ERβ1, ERβ2, ERβ4 &amp; ERβ5) were quantitated using droplet digital polymerase chain reaction. These markers were explored in association with clinical and treatment variables using descriptive statistics. Results: 44 men were included for a total of 92 cfDNA and 94 cfRNA samples, with a median of 2 (range: 1-6) sequential samples per patient. Two mutations (E380Q &amp; D538G) were detected in both cfDNA and cfRNA samples, and two (L536Q &amp; Y537S) were detected in cfRNA samples only. The ER splice variants were widely detected in cfRNA samples, including in non-cancer controls. ERα-36 was detected at lower levels than the other splice variants investigated (p &lt; 0.0001). Despite ubiquitous detection, the levels of splice variants altered consistently with analysis of the following clinical variables: during castration therapy; following castration resistance; and with number of previous treatments. No clear patterns of expression were observed for ER mutants. Serial sample analysis associated increased ERα-36 levels with response to docetaxel or abiraterone therapy. ERβ splice variant levels decreased with progression on all studied therapies. Development of E380Q and L536Q mutations was observed in patients with disease progression. Conclusions: We identified circulating ER mutants and splice variants in men with APC. These variants may be associated with previous treatment use, as well as disease progression on castration therapy or other APC treatments. Further work will explore their utility for predicting treatment response.

A multicenter randomized phase II study of nivolumab in combination with gemcitabine/cisplatin or ipilimumab as first-line therapy for patients with advanced unresectable biliary tract cancer.

TPS541 Background: Patients (pts) with advanced biliary tract cancers (BTC) have poor prognosis with a median overall survival (OS) less than 12 months. This randomized, multi-institutional, phase 2, two-arm study is designed to investigate the role of combinational immunotherapy, using nivolumab with chemotherapy (gemcitabine/cisplatin) or as dual immunotherapy (nivolumab and ipilimumab) in pts with advanced BTC. Methods: Key eligibility criteria include histologically confirmed advanced, unresectable biliary adenocarcinoma (intrahepatic or extrahepatic and gallbladder) without prior systemic treatment, measurable disease per RECISTv1.1, ECOG PS 0-1, and absence of autoimmune disease and/or chronic steroid use. Primary objective is to evaluate the progression-free survival (PFS) rate at 6 months. Secondary objectives include evaluation of overall response rate (ORR), median PFS and OS and safety in this patient population. Exploratory objectives include identification of biomarker predictors of response and mechanisms of resistance through serial (before, on and post therapy) biopsies and blood collection, including sequential whole exome/transcriptomic analysis and immune cell subset analysis (tissue and blood). Arm A therapy provides gemcitabine 1000 mg/m2, cisplatin 25 mg/m2 on days 1, 8 with nivolumab 360 mg on day 1 every 3 weeks for 6 months. In the absence of disease progression, pts may continue single agent nivolumab for up to 2 years. Arm B therapy includes nivolumab 240 mg every 2 weeks and ipilimumab 1 mg/kg every 6 weeks for up to 2 years in absence of disease progression. Accrual goal is 32 evaluable pts per arm. Using a null hypothesis value of 59% median PFS at 6 months, and an 80% alternative hypothesis, this ongoing study has &gt; 80% power, with a one-sided alpha of 0.05 to identify treatment efficacy in one or both study arms. Clinical trial information: NCT03101566.

A pilot study of galunisertib (LY2157299) plus stereotactic body radiotherapy (SBRT) in advanced hepatocellular carcinoma (HCC).

TPS528 Background: Hepatocellular carcinoma (HCC) is a common and lethal malignancy with few effective treatment options. Inherently aggressive disease biology combined with the immunosuppressive hepatic microenvironment creates a unique therapeutic challenge. TGF-β, the strongest known immunosuppressive cytokine [1, 2], modulates the hepatic immune response to various antigens and to ionizing radiation [3, 4]. TGF-β is constitutively released by liver cells and plays a key role in the early and late pathogenesis of HCC [5-7] by dampening the local T-cell response to the oncogenic hepatitis B and –C viruses [4, 8-11]. TGF-β is activated by ionizing radiation, where it blocks the effector T-cell response to cellular destruction and the release of tumor-specific antigens [4]. Preclinical data demonstrate that neutralizing TGF-β during radiation therapy effectively generates a CD8+ T-cell response to multiple endogenous tumor antigens [4], thereby generating an in-situ vaccine against a tumor [12-17]. We hypothesize that the combination of TGF-β receptor inhibition plus radiation therapy will produce a potent and clinically effective antitumor immune response against HCC. Methods: We have enrolled 9 of 15 planned patients on study NCT02906397. Eligibility criteria include inoperable HCC, Childs Pugh score of ≤7, and either failure of or refusal to take sorafenib. Patients must be 4 weeks from prior therapy and may not be taking immunosuppressants. Patients with major cardiac disease or abnormalities or a predisposition toward aneurysm development are excluded. Patients receive galunisertib on days 1-14 of 28 day cycles. SBRT will be delivered in a single fraction of 18 Gy between days 15-28 of C1. Pre-treatment and on-treatment biopsies are obtained, as well as serial blood collections for circulating tumor material. Immunologic evaluation will include TCR deep sequencing to track T-cell receptor clones, analysis of serum inflammatory cytokines, analysis of myeloid and B cell activation, multiplex flow cytometry of PBMCs to measure percentages and absolute counts of T-cell subsets and tissue assessment of immune markers. Peripheral and tissue levels of TGF-β will be assessed. Clinical trial information: NCT02906397.

A single arm phase II study of rucaparib maintenance in patients with advanced pancreatic adenocarcinoma and a known deleterious BRCA1, BRCA2 or PALB2 mutation who have achieved stability on platinum therapy.

TPS531 Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a median overall survival of &lt; 1 year. A subset of PDAC is characterized by a homologous recombination deficiency (HRD). The most well-defined patients within this group are those with deleterious mutations in BRCA1, BRCA2 and PALB2. This subset of tumors respond exceptionally well to treatment with platinum agents, leading to durable responses. However, cumulative toxicity can complicate or even prevent continued therapy, and there is an unmet need to establish maintenance strategies for such patients.Prior studies have shown that BRCA1 and BRCA2 associated PDACs respond to PARP inhibitors. Cross-resistance to platinum and PARP inhibitors exists, so initiating PARP inhibitor therapy after the development of platinum-resistance is an inferior approach. In this setting, we have designed a phase II trial of rucaparib as maintenance therapy for patients with deleterious BRCA1, BRCA2 or PALB2 who have sustained stability on platinum-based treatment. Methods: We have enrolled 2 of 42 planned patients on study NCT03140670. Eligibility criteria include inoperable PDAC, a known somatic or germline deleterious mutation in BRCA1, BRCA2 or PALB2 and stability on platinum-based chemotherapy for ≥16 weeks. Patients who have progressed on platinum-based treatment or who have received prior therapy with PARP inhibitors are excluded. Patients will receive oral rucaparib twice daily continuously in 28-day cycles. The primary endpoint is progression-free survival at 6 months. Secondary endpoints include response rate, duration of response and overall survival. A pre-treatment tumor biopsy and biopsy at progression will be obtained, as well as serial blood collections for circulating tumor material. Correlative assays will include tumor and circulating tumor DNA sequencing to identify genomic predictors of outcome and study resistance mechanisms. Clinical trial information: NCT03140670.

A prospective biomarker trial of microbiome, immune, and genomic markers as predictors of radiation response in anal squamous cell carcinoma.

TPS869 Background: Preclinical evidence suggests that the microbiome plays a significant role in carcinogenesis and in response to immunotherapy and chemotherapy, but little is known about the role of the microbiome in response to radiation. Our group previously demonstrated that in patients with cervical squamous cell carcinoma (SCC), radiation response and immune response to radiation are improved with increased rectal microbial diversity. Methods: We are currently enrolling patients with local anal SCC undergoing definitive chemoradiation on a prospective biomarker trial evaluating changes in the gastrointestinal microbiome, immune microenvironment, and whole genome sequencing using a novel, non-invasive swab biopsy technique in addition to serial blood collections. This biopsy technique allows novel evaluation of dynamic changes throughout the course of chemoradiation. Microbiome sequencing is performed using 16s rRNA sequencing. Immune profiling is done using multiparametric flow cytometry for T-cell activation and exhaustion phenotypes and T-cell receptor sequencing. Inclusion criteria are biopsy proven HPV-related SCC of the anus, with no evidence of metastatic disease, and undergoing definitive chemoradiation. Our population is uniquely treated with cisplatin/5-FU based chemotherapy, rather than lymphotoxic mitomycin-C, allowing unique evaluation of immune populations. The primary endpoint of this study is complete tumor response based on pelvic MRI and digital rectal exam at three weeks. Secondary endpoints include FDG-avid diseases at 3 months and disease recurrence. To date we have accrued 10 patients out a planned 30 patient pilot cohort. We expect the remainder of these patients to be accrued by December 2017.

Identification of mechanisms of resistance to treatment with abiraterone acetate or enzalutamide in patients with castration-resistant prostate cancer (CRPC).

Two androgen receptor (AR)-targeted therapies, enzalutamide and abiraterone acetate plus prednisone (abiraterone), have been approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). Many patients respond to these agents, but both de novo and acquired resistance are common. The authors characterized resistant phenotypes that emerge after treatment with abiraterone or enzalutamide. Patients who received abiraterone or enzalutamide in the course of routine clinical care were consented for serial blood collection. A proprietary system (CellSearch) was used to enumerate and enrich circulating tumor cells (CTCs). RNA-sequencing (RNA-seq) was performed on pools of up to 10 epithelial cell adhesion molecule (EpCAM)-positive/CD45-negative CTCs. The impact of gene expression changes observed in CTCs between patients who responded or were resistant to abiraterone/enzalutamide therapies was further explored in a model cell line system. RNA-seq data from CTCs identified mutations commonly associated with CRPC as well as novel mutations, including several in the ligand-binding domain of AR that could facilitate escape from AR-targeted agents. Ingenuity pathway analysis of differentially regulated genes identified the transforming growth factor β (TGFβ) and cyclin D1 (CCND1) signaling pathways as significantly upregulated in drug-resistant CTCs. Transfection experiments using enzalutamide-sensitive and enzalutamide-resistant LNCaP cells confirmed the involvement of SMAD family member 3, a key mediator of the TGFβ pathway, and of CCND1 in resistance to enzalutamide treatment. The current results indicate that RNA-seq of CTCs representing abiraterone and enzalutamide sensitive and resistant states can identify potential mechanisms of resistance. Therapies targeting the downstream signaling mediated by SMAD family member 3 (SMAD3) and CCND1, such as cyclin-dependent kinase 4/cyclin-dependent kinase 6 inhibitors, could provide new therapeutic options for the treatment of antiandrogen-resistant disease. Cancer 2018;124:1216-24. © 2017 American Cancer Society.

An innovative approach for serial injection in marginal vein and blood collection from auricular artery in New Zealand white Rabbit

Serial injection into marginal vein and blood collection through auricular artery in New Zealand white Rabbit (Oryctolagus cuniculus) is an important procedure for various types of experimental studies. Limitations of the existing methods for serial injection and blood collection includes complex procedures, causes considerable discomfort to rabbits, whole blood samples obtained are highly prone to hemolysis and lastly detailed protocol is not available in the literature.Approximately 10min before commencement of the experiment, a local anesthetic cream was applied over the right and left ear lobes. The skin at the site of sample collection and injection was prepared by shaving the area on both ears and wiping it with alcohol swab. Once prepared, a 26 GA (BD Neoflon) intravenous cannula was inserted into the marginal vein of the ear and secured with an adhesive plaster. In the other ear, a 24 GA (BD Neoflon) intravenous cannula was placed in the auricular/central artery and secured with an adhesive plaster.The novel and refined method described here has been standardized and found to be reliable. The samples obtained using this method is not susceptible to hemolysis and hence we recommend this method for serial injection and blood collection in rabbits.•Easy to perform•Not prone to hemolysis•Detailed methodology described

Glucocorticoid Fast Feedback Inhibition of Stress-Induced ACTH Secretion in the Male Rat: Rate Independence and Stress-State Resistance.

Normal glucocorticoid secretion is critical for physiological and mental health. Glucocorticoid secretion is dynamically regulated by glucocorticoid-negative feedback; however, the mechanisms of that feedback process are poorly understood. We assessed the temporal characteristics of glucocorticoid-negative feedback in vivo using a procedure for drug infusions and serial blood collection in unanesthetized rats that produced a minimal disruption of basal ACTH plasma levels. We compared the negative feedback effectiveness present when stress onset coincides with corticosterone's (CORT) rapidly rising phase (30 sec pretreatment), high plateau phase (15 min pretreatment), or restored basal phase (60 min pretreatment) as well as effectiveness when CORT infusion occurs after the onset of stress (5 min poststress onset). CORT treatment prior to stress onset acted remarkably fast (within 30 sec) to suppress stress-induced ACTH secretion. Furthermore, fast feedback induction did not require rapid increases in CORT at the time of stress onset (hormone rate independent), and those feedback actions were relatively long lasting (≥15 min). In contrast, CORT elevation after stress onset produced limited and delayed ACTH suppression (stress state resistance). There was a parallel stress-state resistance for CORT inhibition of stress-induced Crh heteronuclear RNA in the paraventricular nucleus but not Pomc heteronuclear RNA in the anterior pituitary. CORT treatment did not suppress stress-induced prolactin secretion, suggesting that CORT feedback is restricted to the control of hypothalamic-pituitary-adrenal axis elements of a stress response. These temporal, stress-state, and system-level features of in vivo CORT feedback provide an important physiological context for ex vivo studies of molecular and cellular mechanisms of CORT-negative feedback.

Molecular Markers in Peripheral Blood of Patient with Acute Myocardial Infarction

AIms: Cyclooxygenases (COX) are involved in inflammation and in prostaglandin metabolism. It is estimated that the use of blockers causes a steep rise in cardiovascular events when compared with patients who did not use them. The aim of this report is to show the association of the increase in COX-2 expression in relation to time in a patient with acute myocardial infarction (AMI). Methods:A 54-year-old black woman, hypertensive and ex-cocaine user, presented with typical precordial pain and ischemia in the anterior wall of the myocardium identified by an ECG. Serial blood collections were performed in order to evaluate COX-2 and NF-kB gene expressions. Forty days prior the event she had been submitted to percutaneous coronary angioplasty due to AMI. The coronarIography revealed stent thrombosis.Results: COX-2 and NF-kB expressions peaked in the first 24 hours after the thrombotic event. Discussion: There is an increase in COX-2 expression on atherosclerotic plaques. The use of COX-2 blockers increase the risk of ischemia given their vasoconstrictor effect, resulting in a decrease in oxygen supply to the myocardium. In sum, the increase in COX-2 expression was associated with acute coronary event in this patient.

Biological effects of dietary probiotics on blood characteristics in Hanwoo heifers subjected to lipopolysaccharide (LPS) challenge

This study hypothesized that dietary feed additive containing probiotics alter either immune-related serum substances or serum metabolites in Hanwoo heifers. A probiotic treatment was given at 0.5% top-dressing of concentrate diet for 6 months. The change of immunological indicators in the blood was analyzed under LPS (Lipopolysaccharide) challenge. One day before administration of LPS, all heifers were fitted with an indwelling jugular vein catheter for serial blood collections. Both a serum tube and an EDTA-coated tube were collected at 30-min intervals from - 2 to 8 hours relative to the LPS challenge at time 0 (1 ㎍/㎏ of BW). Serum was used for analyzing albumin (ALB), glucose (GLU), total protein (TP), triglycerides (TG), phosphorus (IP), and non-esterified fatty acids (NEFA). Plasma was used for analyzing white blood cell (WBC), red blood cell (RBC), platelet (PLT) and inflammation-related factors (NE, LY, MO, EO, BA, Hb, HCT, MCV, MCH, MCHC, RDW, MPV). There were significant differences in ALB, GLU, TG, IP, and NEFA concentration with the passage of hours post challenge (p 0.05). The level of WBC, EO, LY, and MO were reduced after LPS challenge (p > 0.05). In conclusion, LPS challenge after dietary supplementation of probiotics changed the levels of both serum metabolites and inflammation-related factors. The increase of GLU and TG indicated a probiotics-positive response under LPS challenge (p < 0.05).

Assessment of blood changes post-challenge with Corynebacterium pseudotuberculosis and its exotoxin (phospholipase D): A comprehensive study in goat.

Aim:There is very little information regarding blood changes during the challenge of phospholipase D (PLD) in goats. Therefore, this experiment was conducted to study the changes in blood after the challenge with Corynebacterium pseudotuberculosis and its exotoxin, PLD to fill in the gap of caseous lymphadenitis (CLA) research.Materials and Methods:Twenty-six crossbred Boer goats aged 12-14 months were divided into 3 groups; the first group n=6 was inoculated with 1 ml phosphate buffered solution s.c. as the control. The second group n=10 was inoculated with C. pseudotuberculosis 1 × 109 cfu s.c. The third group n=10 was intravenous injected with PLD 1 ml/20 kg body weight. Serial blood collections were done at 1 h, 3 h, 5 h, 8 h, and 12 h then every 24 h post-inoculation for the first 30 days of the experiment. Subsequently, the blood collection continued twice a week till the end of the experiment (90 days post-challenge).Results:Both C. pseudotuberculosis and PLD treated groups showed significant changes (p<0.05) in red blood cell count, hemoglobin (Hb), packed cell volume, mean corpuscular volume, mean corpuscular Hb concentration, white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, globulin, and total plasma proteins. Similarly, both treated groups showed significant changes (p<0.05) in alanine transaminase, alkaline phosphatase, aspartate transaminase, total bilirubin, calcium concentration, creatine phosphokinase, creatinine, gamma-glutamyl transpeptidase, urea concentration, lactate dehydrogenase, prothrombin time, and activated partial thromboplastin time.Conclusion:It concluded that C. pseudotuberculosis and PLD have a negative impact on the goat’s health in general reflected by all those changes recorded in the hemogram, leukogram, and the blood chemistry.

A dose-response study of consuming high-fructose corn syrup–sweetened beverages on lipid/lipoprotein risk factors for cardiovascular disease in young adults

National Health and Nutrition Examination Survey data show an increased risk of cardiovascular disease (CVD) mortality with an increased intake of added sugar. We determined the dose-response effects of consuming beverages sweetened with high-fructose corn syrup (HFCS) at zero, low, medium, and high proportions of energy requirements (Ereq) on circulating lipid/lipoprotein risk factors for CVD and uric acid in adults [age: 18-40 y; body mass index (in kg/m(2)): 18-35]. We conducted a parallel-arm, nonrandomized, double-blinded intervention study in which adults participated in 3.5 inpatient days of baseline testing at the University of California Davis Clinical and Translational Science Center's Clinical Research Center. Participants then consumed beverages sweetened with HFCS at 0% (aspartame sweetened, n = 23), 10% (n = 18), 17.5% (n = 16), or 25% (n = 28) of Ereq during 13 outpatient days and during 3.5 inpatient days of intervention testing at the research center. We conducted 24-h serial blood collections during the baseline and intervention testing periods. Consuming beverages containing 10%, 17.5%, or 25% Ereq from HFCS produced significant linear dose-response increases of lipid/lipoprotein risk factors for CVD and uric acid: postprandial triglyceride (0%: 0 ± 4; 10%: 22 ± 8; 17.5%: 25 ± 5: 25%: 37 ± 5 mg/dL, mean of Δ ± SE, P < 0.0001 effect of HFCS-dose), fasting LDL cholesterol (0%: -1.0 ± 3.1; 10%: 7.4 ± 3.2; 17.5%: 8.2 ± 3.1; 25%: 15.9 ± 3.1 mg/dL, P < 0.0001), and 24-h mean uric acid concentrations (0%: -0.13 ± 0.07; 10%: 0.15 ± 0.06; 17.5%: 0.30 ± 0.07; 25%: 0.59 ± 0.09 mg/dL, P < 0.0001). Compared with beverages containing 0% HFCS, all 3 doses of HFCS-containing beverages increased concentrations of postprandial triglyceride, and the 2 higher doses increased fasting and/or postprandial concentrations of non-HDL cholesterol, LDL cholesterol, apolipoprotein B, apolipoprotein CIII, and uric acid. Consuming beverages containing 10%, 17.5%, or 25% Ereq from HFCS produced dose-dependent increases in circulating lipid/lipoprotein risk factors for CVD and uric acid within 2 wk. These results provide mechanistic support for the epidemiologic evidence that the risk of cardiovascular mortality is positively associated with consumption of increasing amounts of added sugars. This trial was registered at clinicaltrials.gov as NCT01103921.

Abstract C203: BRAF and KRAS mutation testing in cell-free DNA and circulating tumor cells from blood of patients with metastatic cancers.

Abstract Background: Cell-free (cf) DNA and circulating tumor cells (CTC) in the blood of cancer patients offer an easily obtainable, low-risk, inexpensive, and repeatedly available source of biologic material for mutation analysis and monitoring of molecular changes throughout cancer therapy. Methods: DNAs from plasma and CTC from patients with advanced cancers who progressed on systemic therapy were tested for BRAF V600 and KRAS G12/G13 mutations using the ICE-COLD-PCR platform. ICE COLD-PCR, "Improved and Complete Enrichment COamplification at Lower Denaturation” selectively amplifies mutant DNA by exploiting differences in denaturation temperatures between mutant DNA duplexes and normal “wild-type” DNA duplexes. KRAS Exon 2 and BRAF Exon 15 ICE COLD-PCR was performed on plasma samples, and from matched CTCs collected using ScreenCell® MB kits (ScreenCell, Sarcelles, France). Amplicons were analyzed by Sanger sequencing methods and results were compared to the mutation status of the archival primary or metastatic tumor tissue as determined in a CLIA-certified lab. Results: Blood samples from 59 patients with advanced cancers (colorectal cancer, n=32; melanoma, n=12; non-small cell lung cancer, n=7; other cancers, n=8), were obtained before treatment and, if possible, sequentially during therapy and tested for BRAF (30), KRAS (29) or BRAF and KRAS (1) mutations. BRAF mutations were detected in 97% (30/31) of archival tumor samples compared to 65% (20/31) of cfDNA samples (agreement 68%) and to 3% (1/31) of CTC samples (agreement 6%). KRAS mutations were detected in 90% (26/29) of archival tumor samples compared to 86% (25/29) of cfDNA samples (agreement 83%) and to 10% (3/29) of CTC samples (agreement 21%); however, CTCs had different KRAS mutation subtypes than those in tumor tissue. Of interest, in 3 patients with serial blood collection for cfDNA BRAF mutations, 2 (melanoma, Erdheim-Chester disease) had BRAF V600E cfDNA mutations at baseline, which disappeared during the response to subsequent therapy. Another patient (melanoma) did not have a BRAF V600E cfDNA mutation immediately after being taken off BRAF inhibitor therapy due to intolerance, but a BRAF V600E cfDNA mutation emerged when the patient was treated with non-BRAF targeting therapy. In 2 patients with serial blood collections for cfDNA KRAS mutations, 2 (colorectal cancer) did not have KRAS cfDNA mutations at baseline, but KRAS cfDNA mutations (G13D, G12D, respectively) emerged following disease progression and subsequent therapy. Conclusions: Detection of BRAF and KRAS mutations in cfDNA can provide a fast and noninvasive alternative to mutation testing in tumor tissue. Although these mutations can be also detected in CTC, the level of concordance with tumor tissue results is low. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C203. Citation Format: Filip Janku, Ben Legendre, Katherine Richardson, Gerald S. Falchook, Aung Naing, Veronica R. Holley, David S. Hong, Ralph G. Zinner, Siqing Fu, Apostolia M. Tsimberidou, Vivek Subbiah, Daniel D. Karp, Sarina A. Piha-Paul, Jennifer J. Wheler, Vanda M. Stepanek, Goran Cabrilo, Rajyalakshmi Luthra, Funda Meric-Bernstam, Amy Kruempel, Jaclyn Pope, Courtney Cubrich, Grant Wu, Yanggu Shi, Marcia Lewis, Razelle Kurzrock. BRAF and KRAS mutation testing in cell-free DNA and circulating tumor cells from blood of patients with metastatic cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C203.

A clinical investigation of inhibitory effect of panobinostat on CYP2D6 substrate in patients with advanced cancer

Panobinostat is a potent oral pan-deacetylase inhibitor with promising clinical activity in hematologic malignancies. Panobinostat was shown to inhibit CYP2D6 activity in vitro; thus understanding the magnitude of the potential clinical inhibition of panobinostat on co-medications that are CYP2D6 substrates becomes important. This study evaluated the effects of co-administration of panobinostat with a sensitive CYP2D6 substrate, dextromethorphan (DM), in patients with advanced cancer who have functional CYP2D6 genes. Patients received 60mg DM alone on day 1, panobinostat at 20mg alone on days 3 and 5, and both agents on day 8. Plasma concentrations of DM and its metabolite dextrorphan (DX) were determined by liquid chromatography-tandem mass spectrometry following serial blood collections on day 1 (DM alone) and day 8 (in combination with panobinostat). Panobinostat increased DM exposure by 64% [geometric mean ratio (GMR), 1.64 (90% confidence interval (CI), 1.17-2.31)] and DX exposure by 29% (GMR, 1.29 [90% CI, 1.10-1.51]). These results indicated that panobinostat weakly inhibited a sensitive CYP2D6 substrate in cancer patients by increasing DM exposure by less than twofold. Safety monitoring of sensitive CYP2D6 substrates with narrow therapeutic index is recommended when co-administering with panobinostat in future clinical practice.

Biological rhythms related to metabolism in Japanese Shorthorn cattle under varying environments and management techniques

Plasma insulin (INS), thyroxin (T4 ), glucose (GLU), non-esterified fatty acid (NEFA), blood urea nitrogen (BUN), rectal temperature (RT) and eating behavior were evaluated in Japanese Shorthorn cattle under varying external environments and management techniques. Serial blood collection and assessments of RT and eating behavior were performed over 48 h in the spring, summer, autumn and winter in four female cattle reared under either free-stall and ad libitum feeding (FA) conditions or tie-stall and restricted feeding (TR) conditions. Cycle patterns for each parameter were examined using spectral analysis, and correlations between parameters were investigated using cross-spectral analysis. Rhythms for all parameters, except eating behavior and T4 , did not differ significantly among the varied external environments and between management techniques, although seasonal differences in the concentration or value of parameters were observed. An approximate 3- or 4-h rhythm cycle detected in T4 , GLU, NEFA, BUN, and RT might be the common metabolic rhythm. Under both conditions, the metabolite levels showed strong correlations with eating behavior. Moreover, GLU positively correlated with INS at lag time of 0 h, as did eating behavior and RT.

Monitoring of benzene-induced hematotoxicity in mice by serial leukocyte counting using a microcavity array

Monitoring of hematotoxicity, which requires serial blood collection, is difficult to carry out in small animals due to a lack of non-invasive, individual animal-appropriate techniques that enable enumeration of leukocyte subsets from limited amounts of whole blood. In this study, a microfluidic device equipped with a microcavity array that enables highly efficient separation of leukocytes from submicroliters of whole blood was applied for hematotoxicity monitoring in mice. The microcavity array can specifically separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Mouse leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered almost 100% of mouse leukocytes in 0.1μL of whole blood without the effect of serial blood collection such as changes in body weight and total leukocyte count. We assessed benzene-associated hematotoxicity in mice using this system. Mice were administered with benzene once daily and the depression of leukocyte numbers induced in individual mice was successfully monitored from tail vein blood collected every other day for 2 weeks. Serial monitoring of the leukocyte number in individual mice will contribute to the understanding of hematotoxicity and reduction of the number of animal experiment trials.

Abstract B24: De novo and acquired resistance to first-line standard therapy in colorectal cancer: from cell lines to metastatic tumors

Abstract Introduction: Personalized medicine (PM) is a concept that has raised high expectations amongst scientists, clinicians, and patients. An emerging approach is to examine tumor biopsy material for genomic changes that are known targets of currently available therapeutic agents, with the assumption that a clinical benefit will be observed if the target is inhibited. While striking anecdotal reports are predictable from this approach, the clinical impact of these agents is limited by the inevitable development of therapeutic resistance. Our focus is on the design of parallel research programs using both in vitro and in vivo strategies, in an effort to delay or inhibit resistance. We present here preliminary data for a signature of resistance to standard first-line treatment - fluorouracil, folinic acid, oxaliplatin and bevacizumab (FOLFOX/B) using cell line models of resistance to this regimen. In parallel, we are conducting a prospective study to identify biomarkers of clinical resistance to first-line therapy in patients with metastatic colorectal cancer (CRC) (NCT00984048). Methods: Ten established CRC cell lines were treated with FOLFOX/B and categorized as resistant or sensitive based on IC50 values. In parallel, patients who consented to an initial biopsy and one at disease progression following an initial response were identified as intrinsically resistant or as having acquired resistance during treatment. CRC cell lines that were initially sensitive were rendered resistant to mimic the acquired resistance in patients, by serial passages with gradual increases in concentration of the combination regimen. We compared microarray data from three sensitive and three resistant cell lines. Results: We found a different expression pattern from microarray data comparing sensitive and resistant cell lines, thereby indicating a potential signature of resistance to FOLFOX/B. Interestingly, we found that the Src family kinase Lyn was overexpressed in resistant cells lines. Treating cells with non-cytotoxic concentration of dasatinib, a dual Src family kinase and Abl inhibitor, sensitized both the parental sensitive cells and the cells with acquired resistance to FOLFOX/B, thereby suggesting that combination treatment with dasatinib may be effective in delaying or inhibiting resistance. We have thus far collected needle core biopsies from liver metastases from forty patients who agreed to partake in this multi-center trial. Eligible patients have confirmed metastatic CRC, measurable disease, and consent to three needle-core biopsies (NCBs) of a non-resectable liver metastasis before treatment and at resistance, as well as serial blood collection throughout the study. Using standard operating procedures developed for this trial, we were able to both preserve morphology and obtain high-quality genomic material from biopsy tissue. We will determine if the resistance signature and overexpression of Lyn observed in the resistant CRC cell lines are similarly demonstrated in patients that were intrinsically resistant to FOLFOX/B. Conclusions: We have designed parallel in vitro and in vivo experiments to study resistance to standard first-line treatment for mCRC. These studies provide insight on metastatic signatures of resistance and suggest combination therapies to delay or inhibit therapeutic resistance in patients.