bluuebunny/crossref_metadata_2025 huggingface dataset:
Screening and Triage Test for Early Detection of Gastric Cancer (STEAD-GC): A noninvasive trial for early detection of gastric cancer.
Authors: Corvalan Alejandro , Maturana Maria Jose , Sanchez Marianela , Calvo Alfonso , Ferreccio Catterina
Published: February 2017

17

Background: Gastric cancer (GC) is the second leading cause of cancer-related deaths worldwide. Previously, we identified a potential biomarker for non-invasive detection of GC, the DNA methylation of the promoter region of Reprimo, a p53-dependent G2 arrest mediator candidate (Clin Cancer Res 2008;14:6264-9). Furthermore, we developed a quantitative assay (MethyLight) for a mass screening of GC (DDW2011-1029128). Here we reported the preliminary findings of our ongoing prospective trial STEAD-GC (Screening and Triage test for Early Detection of Gastric Cancer) which is being conducted in Chile, a country with a high mortality rate for GC. Methods: Twenty GC cases (tumor, non-tumor tissues and plasma samples) and 41 symptomatic chronic gastritis cases (29 tissues and 12 pairs of tissue and plasma samples) were evaluated for Reprimo levels by MethyLight after DNA extraction and bisulfite conversion. Results: Concentrations of DNA were similar in both groups (Avg 32.2 ng/ml [range: 8.9-70.8 ng/ml]). The average DNA levels of Reprimo were higher in GC [964,215 copies/ml, 539,593 copies/ml and 80,113 copies/ml in tumor, non-tumor and plasma, respectively] but lower in symptomatic chronic gastritis [137,721 copies/ml and 8,387 copies/ml, tissue and plasma, respectively]. Methods: Twenty GC cases (tumor, non-tumor tissues, and plasma samples) and 41 symptomatic chronic gastritis cases (29 tissues and 12 pairs of tissue and plasma samples) were evaluated for Reprimo levels by MethyLight after DNA extraction and bisulfite conversion. Conclusions: By using our previous cut-off of 15,125 copies/ml, our method correctly identified 10 out of 12 gastritis cases and 16 out of 20 GC cases (p value <0.001). Our data confirms our non-invasive method for early detection of GC may be suitable for a mass screening of GC.
CD98hc expression to predict prognosis in renal cell cancer.
Authors: Prager Gerald , Poettler Marina
Published: February 2017

26

Background: CD98, a transmembrane protein, has a heteromeric structure, consisting of a heavy subunit (CD98hc) and a light subunit, extracellular linked together via disulfid bounds. A genetic knockout of CD98hc is embryonic lethal and overexpression of CD98hc in somatic cells led to malignant transformation. CD98hc is highly expressed in low differentiated papillary, clear cell and chromophobe RCC, but not in benign tumors. Notably, CD98hc expression directly correlated with grade of differentiation. Methods: To evaluate a potential functional role of CD98hc in renal cancer cell metastatic behavior, we generated a stable low CD98hc clear cell RCC cell line (Caki2) via lentiviral shRNA infection and compared tumor cell behavior with a high expressing mock transfected control. Results: We found that tumor cell behavior such as proliferation (52 ± 3% less 3[H] thymidin – incorporation in low CD98hc/Caki2 cells), cell survival upon anoikis (46% late and 45% early apoptosis in low CD98hc/Caki2 cells compared to high/CD98hc/Caki2 cells with 18% late apoptosis and 64% early apoptosis) and invasion/transmigration (520 ± 67 cells / field after 24h in low CD98hc/ Caki2 cells and 1257 ± 346 cells / field in high CD98hc/Caki2 cells analyzed in a modified boyden chamber) were considerably impaired whenever CD98hc expression was downregulated. To examine the mechanism by which CD98hc affected metastatic tumor cell behavior, we introduced two mutations in a reconstitution (silent mutation). A truncation mutant interfered with integrin interaction and a two point mutations (Cys109Ser and Cys330Ser) mutant affected amino acid transporter activity. Whenever integrin/CD98hc interaction was impaired tumor cell behavior including cell proliferation, cell survival, invasio/transmigration, and cell spreading as well as signal transduction pathways including FAK, c-src, MEK/ERK pathways were significantly compromised resembling the low CD98hc phenotype. Conclusions: For these data we conclude that metastatic tumor cell behavior such as cell survival, invasion and proliferation are dependent on CD98hc expression in renal cancer cells.
A pilot study on the anticancer effects of novel candidate agent COH-SR4 in lung cancer.
Authors: Berz David
Published: February 2017

28

Background: Non-small cell lung cancer is the leading cause of cancer deaths in the US. The success of current treatment strategies is limited by frequent aberrations in multiple signaling pathways. This includes loss-of-function mutations in the tumor suppressor p53 and activating mutations in multiple growth factor receptors, which converge to activate PI3K/Akt pathway. This calls for the development of novel, more active treatments. We investigated the anti-cancer effects of a novel compound called 1, 3 bis (3, 5-dichlorophenyl) urea (COH-SR4) in lung cancer. Methods: The anticancer effects of COH-SR4 were tested in p53-null H358 lung cancer cells. The antiproliferative effects were investigated in vitro by MTT assay. The bio-availability and antitumor effects were determined in vivo following the administration of 4 mg/kg of COH-SR4 to mice and H358-nu/nu nude mice xenografts. Results: The treatment with COH-SR4 resulted in effective inhibition of the proliferative potential of H358 lung cancer cells [IC50: 23+2 µM], effectively inducing apoptosis. The 4 mg/kg COH-SR4 administration resulted in a free serum concentration of 1+ 0.3 µM. Regression of established H358-xenografts was achieved without any overt toxicity. The histopathology of resected tumor sections revealed an increase in pAMPK (T172) and a decrease in the nuclear proliferative marker Ki 67 and angiogenesis marker CD31. Western blot analyses of resected tumor lysates revealed a decrease in pAkt (S473) and anti-apoptotic protein Bcl2 along with an increase in pAMPK (T172), pro-apoptotic Bax and cleaved PARP levels. In addition, COH-SR4 lead to a decrease in the levels of the cell cycle regulators CDK4 and cyclin B1 as well as the mesenchymal marker vimentin, whilst increasing the epithelial marker E-cadherin. Conclusions: COH-SR4 represents a novel agent for the treatment of non small cell lung cancer. We demonstrated pronounced anti-proliferative and pro-apoptotic activity in vitro and in vivo as well as the capability to promote physiologic, epithelial differentiation. This implies not only therapeutic, but also preventive potential. Further studies are needed to establish the best possible clinical applications of COH-SR4 in lung cancer.
Improving risk stratification in superficial (T1) gastroesophageal adenocarcinoma: Association of the intestinal phenotype (MUC2 positive) with prognosis.
Authors: Davison Jon M. , Ellis Shane , Foxwell Tyler , Zaidi Ali H , Jobe Blair A , Gibson Michael K. , Kuan Shih-Fan , Nason Katie Sue
Published: February 2017

33

Background: An increasing proportion of esophageal and gastroesophageal junction adenocarcinomas (EAC) are limited to the superficial mucosal layer (stage classification T1) at the time of diagnosis. Intramucosal lesions (stage classification T1a) are increasingly managed with endoscopic resection due to their relatively low risk of nodal metastasis and favorable prognosis compared to those with submucosal invasion (stage classification T1b). At esophagectomy, however, the majority of T1b EAC are node-negative; conversely, a small proportion of T1a EAC will have nodal metastasis at esophagectomy and/or develop recurrent disease. Additional prognostication tools, such as tumor biomarker panels, are needed to optimize treatment algorithms. Known to carry prognostic significance in other tumors, we evaluated mucin protein expression to determine whether the mucin phenotype in early EAC was associated with survival outcomes. Methods: We classified 141 induction naive, resected, T1 EAC as intestinal-, gastric- or ductal-type depending on the expression pattern of gastrointestinal mucins (MUC2, MUC5AC, MUC6 and MUC1) determined by immunohistochemistry (IHC) on tissue microarrays. CDX2 expression was assessed by IHC and EGFR amplification was assessed by in situ hybridization. The association between survival and mucin subtypes was determined in univariate and multivariate analyses. Results: The intestinal phenotype (MUC2+) is associated with poor prognosis in T1b (HR 2.1, 95%CI 1.1-4.0) and node negative T1 EAC (HR 2.5, 95%CI 1.1-5.7) after adjusting for other significant prognostic factors. Intestinal type EAC also showed accelerated time to first recurrence in these subgroups. The colloid histologic subtype and CDX2 expression were positively associated with MUC2 expression, but EGFR amplification was not. Conclusions: The mucin phenotype may serve as a useful adjunct to conventional risk assessment for optimizing therapy for potentially resectable, superficial EAC.
A phase II trial design with direct assignment option for initial marker validation.
Authors: Mandrekar Sumithra J. , An Ming-Wen , Sargent Daniel J.
Published: February 2017

34

Background: Phase II clinical trials aim to identify promising experimental regimens for further testing in phase III trials. Testing targeted therapies with predictive biomarkers mandates efficient trial designs. Current biomarker-based trial designs, including the enrichment, all-comers, and adaptive designs, randomize patients to receive treatment or not throughout the entire duration of the trial. Recognizing the need for randomization yet acknowledging the possibility of promising but nonconclusive results after a preplanned interim analysis (IA), we propose a two-stage phase II design that allows for the possibility of direct assignment (i.e., stop randomization and assign all patients to the experimental arm in stage II) based on IA results. Methods: Using simulations, we compared properties of the direct assignment option design to a 1:1 randomized phase II design and assessed the impact of the timing of IA (after 33%, 50%, or 67% of accrual) and number of IA (one versus two with option for direct assignment at the first and second) over a range of response rate ratios (between 1.0 and 3.0). Results: Between 12% and 30% of the trials (out of 6,000 simulated trials) adopt direct assignment in stage II, with direct adoption depending on the treatment effect size and specified type I error rate (TIER). The direct assignment option design has minimal loss in power (<1.8%) and minimal increase in T1ER (<2.1%) compared to a 1:1 randomized design. The maximum loss in power across possible timings of IA was <1.2%. For the direct assignment option design, there was a 20%-50% increase in the number of patients treated on the experimental (vs. control) arm for the 1 IA case, and 40%-100% increase for the 2 IA case. Conclusions: Testing predictive biomarkers in clinical trials requires new design strategies. In the spectrum of phase II designs from adaptive to balanced randomized all-comers or enrichment designs, the direct assignment design provides a middle ground with desirable statistical properties that may appeal to both clinicians and patients.
The prognostic value of seven soluble proteins measured in plasma or serum from patients with colorectal cancer in TNM stages I-III.
Authors: Christensen Ib Jarle , Høyer-Hansen Gunilla , Thurison Tine , Dowell Barry , Johansen Julia S. , Henriksen Rikke , Rohrberg Kristoffer Staal , Brunner Nils , Nielsen Hans J.
Published: February 2017

35

Background: In colorectal cancer (CRC) several independent blood-borne biomarkers have been proposed as prognostic and/or predictive markers. However only CEA is at present recommended as a serological CRC biomarker. We have identified 6 other biomarkers and the aim of this study is to see if a combination of markers improves the prognostic and/or predictive value. Methods: Two-hundred and twenty-eight patients with CRC have been included in this study, all with TNM stages I-III. Overall survival (OS) was chosen as the primary endpoint with 93 events and the minimum follow-up was 47 months. Seven biomarkers measured in plasma or serum were analysed: CEA, TIMP-1, the 3 soluble uPAR forms suPAR(I-III), suPAR(I-III)+(II-III) and uPAR(I), PAI-1 and YKL-40. Multivariable analyses of OS were done using regression analysis and results presented by 3 and 5 year OS rates with area under the receiver operating characteristic curve (AUC). All biomarker levels were analysed on the log scale (base 2). Results: High levels of each of the included biomarker were significantly associated in a multivariable analysis (adjusted for age, gender, TNM stage, tumor localization, adjuvant chemotherapy,interaction between adjuvant therapy and biomarker) to poor prognosis in patients not receiving adjuvant chemotherapy. The results are shown for those patients (see Table). The uPAR forms were the only biomarkers significantly associated to OS in patients receiving adjuvant chemotherapy. Combining the markers resulted in an enhanced prediction of 3 and 5 year OS. For TNM stage II patients not receiving adjuvant therapy, the AUC was 0.765 for 3 year OS and 0.745 for 5 year OS. The corresponding AUC’s for TNM stage III were 0.859 and 0.861. Conclusions: The presented combination of blood biomarkers are shown to predict OS with higher precision than any single marker for patients not receiving adjuvant chemotherapy and may provide useful information for use in the management of patients with CRC. [Table: see text]
Targeted deep sequencing as a diagnostic and research method for detecting EGFR and ALK pathway driver genes in FFPE tissue of lung and colorectal carcinomas.
Authors: Knuutila Sakari , Merkkiniemi Katja , Rönty Mikko , Wirtanen Aino , Remes Satu Maria , Bloor Stuart , Salmenkivi Kaisa , Knuuttila Aija , Sarhadi Virinder Kaur
Published: February 2017

39

Background: Molecular targeted tyrosine kinase inhibitor (TKI) treatments have made it crucial to perform diagnostic tests of multiple molecular targets. In lung carcinoma there are close to ten clinically relevant gene mutations, copy number alterations and/or gene fusions, such as ALK, EGFR, ERBB2, KRAS, BRAF, MET, PTEN, PI3KCA, ROS1 and RET. Presently, several different tests are utilized, requiring a high amount of tumor material and long turnaround time. Next generation sequencing or targeted deep sequencing (TDS) has opened a new era for rapid genome-wide analyses of mutations, copy number alterations and gene fusions. Our aimwas to 1) prove feasibility for applying TDS to FFPE samples, 2) compare mutations detected by prevalent methods & TDS, and 3) mine novel clinically and biologically relevant genes in lung and colorectal carcinoma. Methods: For TDS, we selected 192 lung carcinoma and colorectal carcinoma related genes and microRNA genes, focusing on the EGFR and ALK pathways. In total, 98 FFPE specimens were studied. Agilent SureSelect system and Illumina sequencing was adopted for the analysis. For diagnostic validation the following genes were selected: EGFR, KRAS, BRAF, PTEN, PI3K, RET and ALK. TDS results were confirmed by PCR, FISH and IHC. Results: We focused on the genes selected for diagnostic validation. Successful results were obtained from all specimens. The results from TDS correlated significantly with those obtained from PCR, FISH, and IHC. Importantly, TDS revealed novel mutations not detected by targeted PCR. Conclusions: An enormous advantage of TDS is that multiple mutation screening can be achieved in one analysis (saving time and material), and most importantly, provides enormous amounts of novel information, for example understanding mechanisms for drug resistance. This study was supported by Finnish Academy, Sigrid Jusélius Foundation, Finnish Cancer Organizations, the special governmental subsidy research funds appropriated to the Helsinki and Uusimaa Hospital District (HUS EVO), Pfizer Oy, AstraZeneca AS, Lab21 Ltd, Abbott Molecular Inc.
Development and validation of a digital Gleason score biomarker signature for risk stratification of patients with prostate cancer.
Authors: Erho Nicholas , Vergara Ismael A. , Buerki Christine , Ghadessi Mercedeh , Crisan Anamaria , Sierocinski Thomas , Haddad Zaid , Zimmermann Benedikt , Harko Sebastian , Sosu-Sedzorme Worlanyo , Triche Timothy J. , Jenkins Robert B. , Davicioni Elai
Published: February 2017

40

Background: Gleason score (GS) is the most widely used grading system of cell differentiation in prostate cancer and one of the best pathological predictors of disease progression. Patients with high GS (> 7) are the most likely to experience metastasis, whereas patients with low GS (< 7) are expected to have favorable long-term outcomes. In addition to the subjective nature of GS assessment, patients with GS 7 represent a heterogeneous group in terms of patient outcomes. In this study a biomarker signature is developed and validated which could improve the prediction of high risk disease among GS 7 radical prostatectomy (RP) patients. Methods: Patient specimens from the Mayo Clinic RP Registry (n = 764) were used for feature selection and training of a 392-feature K-nearest neighbor (KNN, k = 11) classifier. These 392 genomic features were identified by assessing differential expression between patients with GS < 7 and those with GS > 7, using a Bonferroni adjusted T-Test with a p-value threshold of 0.05. The classifier was subsequently validated in two independent patient cohorts (Memorial Sloan Kettering [MSKCC] and German Cancer Research Center [DKFZ]) and compared to a state of the art biomarker signature (Penney et al. 2011). Results: In the MSKCC dataset (GSE21034) our model segregated GS < 7 from GS > 7 patients (n = 56) with an area under the receiver operating characteristic curve (AUC) of 0.97, comparable to the AUC of 0.94 obtained by Penney et al. in their independent validation set (n = 45). Strong performance was observed by our model when discriminating between primary Gleason grade (pGG) 3 and pGG 4 & 5 patients in the MSKCC (n = 130) and DKFZ prostate cancer (GSE29079, n = 47) datasets, achieving AUCs of 0.75 and 0.87, respectively. In the challenging GS 7 subset, our model segregated GS 3 + 4 and 4 + 3 patients in the DKFZ dataset (n = 64) with an AUC of 0.81 outperforming the Penney et al. signature (AUC = 0.60). Conclusions: A biomarker signature was developed which discriminates between low and high GS patients, outperforming a previously reported signature. Further validation of this biomarker signature in additional post RP patients, as well as, pretreatment biopsy specimens is warranted.
Association of CD44s and CD44v6 expression with survival outcomes in malignant fibrious histiocytoma.
Authors: Peiper Matthias , Boelke Edwin , Matuschek Christiane , Stoecklein Nikolas H. , Poremba Christopher , Schauer Matthias , Knoefel Wolfram Trudo
Published: February 2017

44

Background: New prognostic markers are being searched to determine the value of adjuvant and/or palliative treatment in soft tissue sarcomas (STS). Several studies showed that CD44 expression was associated with a better outcome in certain cancers, such as bladder tumors. The aim of this study was to evaluate the expression of CD44 in STS of the adult and to determine whether this correlates with the clinical outcome. Methods: We examined the clinical outcome of 34 adult MFH patients (19 males and 15 females, average age 62 years, median 63 years, ranged from 38 to 88 years) who underwent curative treatment The majority of patients received adjuvant radiotherapy (n = 25). No patients received initial adjuvant chemotherapy. The prognostic value of CD44s and isoform expression were evaluated by immunohistochemistry of tissue microarrays. Results: At first, we split the data for each variant of CD44 into two equally sized groups at the medians to the expression values (low and high). We apply a log rank test on the two groups and use the p value as an evaluation criterion for the usefulness of the grouping. CD44s and CD44v6 expression significantly correlated with an improved outcome whereas CD44v8 and h CD44 were not significant. Conclusions: CD44s and CD44v6 expression seem to be associated with improved survival in malignant fibrous histiocytoma.
Development and initial validation of a metabolite profile for the early detection of breast cancer recurrence.
Authors: Raftery Daniel , Gu Haiwei , Asiago Vincent , Alvarado Leiddy , Djukovic Danijel , Ballas Robert
Published: February 2017

5

Background: The detection of recurrent breast cancer is limited by poorly performing CA markers that are both insensitive and late markers. Because of their sensitivity to biological status, metabolite markers may provide better diagnostic performance and earlier detection. Detectable perturbations in the metabolic profiles of patients often result from altered metabolism in cancer. We report on the discovery and initial validation of a metabolite profile for the early detection of recurrent breast cancer. Methods: We applied a combination of nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS) to analyze the metabolite profiles of 116 serial serum samples from 20 recurring patients and 141 serial samples from 36 breast cancer survivors with no evidence of disease (NED). Multivariate analysis was used to identify 11 metabolite markers that were used to build a model with high accuracy (AUROC >0.88 using 10 fold cross validation) with a sensitivity of 68% and specificity of 94%. Strikingly, over 55% of the patients could be correctly predicted to have recurrence on average 13 months before clinical diagnosis, representing a large improvement over the current diagnostic assays CA 27.29 and CA 15-3 (Cancer Res. 2010; 70, 8309-18). The metabolites were then ported to an LC-MS/MS platform and a method was developed to quantify these metabolites using stable isotope labeled compounds. Due to difficulty in obtaining some labeled compounds the profile was reduced to 9 metabolites. The multivariate algorithm was recalculating using five-fold cross validation and using 211 patient samples. During this work we found that the performance of the assay could be improved by adding CA27-29 values to the assay, which mainly ensured excellent specificity. Results: The profile was tested using a separate validation set of 96 patient samples run identically. The performance was similar to the training set with a sensitivity of 65% and specificity of 93%. Recurrence detection was approximately 11 months ahead of clinical diagnosis (based on imaging for symptomatic patients) and about 2 years ahead of CA 27-29 alone. Conclusions: This metabolite profile has promise for early recurrence detection.
Shed alcam as a biomarker for urogenital cancers.
Authors: Arnold Shanna , Jones-Paris Celestial , Hansen Amanda Georgia , Fadare Oluwole , Clark Peter , Zijlstra Andries
Published: February 2017

50

Background: ALCAM (Activated Leukocyte Cell Adhesion Molecule) is thought to be involved in tumor cell adhesion, migration and metastasis. The ectodomain responsible for adhesion is proteolytically shed. Elevated shedding of ALCAM ectodomain is associated with malignant progression. Considering the proximity of urogentical cancer to both urinary secretions and hematogenous circulation we investigated if ALCAM shed into urine and/or blood could act as a prognostic or diagnostic marker of disease and patient outcome. Methods: Shed ALCAM in serum and urine was measured by ELISA while ALCAM shedding in tissues was detected with a unique immunohistochemical approach. ALCAM shedding was correlated with patient parameters including tumor stage, metastasis, recurrence, overall and disease-specific survival. Results: ALCAM shedding was compared across tissue and fluids from normal age-matched healthy adults, non-urological neoplasia, and non-cancer patients. Elevated ALCAM shedding was readily detected in fluids and tissue from patients with urogenital cancers. However, only the detection of shedding in tissue and urine correlated significantly with disease progression and patient outcome. Specifically, shed ALCAM detected in urine collected from bladder cancer patients undergoing cystectomy (Vanderbilt 2000-2010) correlated strongly with patient outcome. Urine ALCAM correlates positively with invasiveness (80%), and is predictive of recurrence as well as survival (HR=10.2 and 2.9). Urinary ALCAM ROC curves show the optimal sensitivity and specificity of predicting recurrence (90 and 68%) and survival (62 and 83%). Conclusions: We show for the first time that cleaved ALCAM is detectable in the urine and that urinary ALCAM is a novel biomarker in urogenital cancers. ALCAM shedding is marker of cancer progression and patient outcome with high specificity and accuracy. Evaluating levels of shed ALCAM in urine offers a unique and effective method to diagnose, assess, and stratify patients with urogenital cancers. We find this correlation particularly exciting because ALCAM is functionally involved in cancer progression and therefore reports on cellular behavior rather than mere presence of neoplastic disease.
DNA mismatch repair deficiency and benefit from adjuvant bevacizumab in NSABP C-08: Molecular profiling results.
Authors: Pogue-Geile Katherine L. , Tanaka Noriko , Gavin Patrick , Yothers Greg , Colangelo Linda H. , Kim Chungyeul , Kim Seong-Rim , Fumagalli Debora , Taniyama Yusuke , Sharif Saima , O'Connell Michael J. , Paik Soonmyung
Published: February 2017

55

Background: The purpose of this study was to identify biomarkers that define a subset of patients who received benefit from bevacizumab (bev) in NSABP trial C-08, even though bev did not improve outcomes over standard adjuvant chemotherapy (CT) in the treatment of stage II and III colon cancer. Methods: A randomly selected cohort of C-08 cases (N=500) were profiled for whole genome expression (N=445) and for mutations (N=463) in KRAS, NRAS, PIK3CA, and MET. BRAF mutations and mismatch repair (MMR) status were profiled on the available cases (N=1,764 and 1,993, respectively). Cox proportional hazard models were used to assess prognosis and prediction for the value of bev using overall survival (0S) and time to recurrence (TTR) as end points. Results: The effect of bev was different for MMR deficient (MMR-d) and proficient tumors for OS (interaction p=.035) but not TTR (interaction p=.08). Patients with MMR-d (N=252) tumors showed a significant benefit from the addition of bev to CT for OS (hazard ratio =0.52 (95% CI: 0.29-0.94, p=0.028). KRAS, NRAS, PIK3CA, and MET were not significant for interaction with bev in the discovery cohort. BRAF mutations were associated with MMR status (p<.0001) and the prognostic value of MMR depended on BRAF for TTR (interaction p=.027) but not OS (interaction p=.31). The effect of bev was independent of BRAF (interaction p=.28 TTR and .37 OS). Three-factor interaction tests for bev, MMR, and BRAF were not significant for either endpoint. Gene expression analysis with BRB array tools identified 5 BioCarta pathways (p<0.05) which differentially expressed in 4 statistical tests; 4 of these pathways were directly or indirectly involved in T cell activation and one was involved in the activation of VEGF. Conclusions: Patients in C-08 with MMR-d tumors received benefit from bev treatment but these results need to be validated in a separate study. Gene expression data suggest that T-cells may be differentially expressed based on MMR status. Activation of VEGF has been shown to suppress T-cell development (Ohm et al. Blood. 2003:10;4878). A speculative possibility for the benefit of bev in MMR-d tumors may be due to blocking of VEGF, releasing T cells from VEGF suppression.
Correlation of oncogenic mutations in circulating cell-free DNA (cfDNA) and tumor tissue through a multiplex sequencing platform in patients under consideration for phase I trials.
Authors: Mateo Joaquin , Ong Michael , Pope Lorna , Mulick Cassidy Amy , Yap Timothy Anthony , Perkins Geraldine , Riisnaes Ruth , Flohr Penelope , Thway Khin , Gonzalez Michael , Saeed Sadiya , Young Anne Mary , Toloui Helen Nicole , Khan Khurum Hayat , Moran Kirsty , Ng Matthew , Molife L Rhoda , Banerji Udai , Kaye Stanley B. , De Bono Johann Sebastian
Published: February 2017

6

Background: Previous studies suggest that tumoral cfDNA is a potential biomarker in cancer. Methods: Prospective collection of samples from patients (pts) referred to a phase I trials unit, in 2 cohorts: “A”(manual isolation of cfDNA, Sep09-Dec10) and “B”(robotic isolation, Jan11-Sep11) and from 41 healthy volunteers (HV). CfDNA and tumor DNA from matched formalin-fixed paraffin-embedded (FFPE) tissue were isolated, quantified and analyzed for 238 oncogenic mutations (OM) in 19 oncogenes using Sequenom OncoCarta Panel 1.0. Mutation data were used for trial allocation when available. Statistical analyses used Pearson’s Chi-squared test and Mann-Whitney test. Results: 280 pts were included: breast (60; 21%), ovarian (44; 16%), and colorectal (42; 15%). 265 (95%) plasma and 194 (69%) FFPE samples were suitable for analysis. In 181 pts (65%) both samples were available for comparison. Median turnover time for cfDNA 7 days (range 5-14). Median [cfDNA] (concentration; ng/ml) in pts was higher than in HV (“A” 21 v 6.4;p<0.01, “B” 34 v 24;p<0.01). Detection of OM in cfDNA was more frequent when [cfDNA] was above the HV range (67% v 33% p=0.002). Among 181 pts with both samples analyzed, 53 OM were detected in cfDNA, with high concordance with tumor analysis (Table), especially for RAS in colorectal tumors (10/11 91%) and PIK3CAin breast (4/4 100%). In contrast, 29 pts (16%) had OM detected in tumor but not in plasma. The rate of concordance between OM detected in plasma v FFPE was independent of [cfDNA]. 154 pts were enrolled in phase I trials and 26 were given a matched drug according to the mutational analysis. Conclusions: cfDNA may be a useful tool to detect OM in a time frame suitable for clinical decision-making. Concordance of OM found in cfDNA with FFPE samples is high; discordance in this study may be explained by limitations of multiplexed-sequencing techniques, tumor heterogeneity or biologic evolution of the disease. [Table: see text]
An explorative analysis of TOP1 copy number alterations in a chemonaive stage III CRC patient cohort.
Authors: Smith David Hersi , Rømer Maria Unni , Jensen Niels Frank , Nygaard Sune Boris , Christensen Ib Jarle , Müller Sven , Nielsen Hans J. , Brunner Nils , Nielsen Kirsten Vang
Published: February 2017

66

Background: Topoisomerase I (TOP1) is the target of TOP1 inhibitor chemotherapy. The TOP1 gene, located at 20q12, is frequently gained in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene alterations in advanced CRC. Methods: Nine CRC cell line metaphase spreads were analyzed with a TOP1 probe in combination with a reference probe covering either the centromeric region of chromosome 20 (CEN20) or chromosome 2 (CEN2). Tissue sections from 154 stage III CRC patients, previously studied with TOP1/CEN20, were analyzed with TOP1/CEN2. Relationships between biomarker status and overall survival (OS), time to recurrence (TTR) in CRC and to local recurrence (LR; rectal cancer only) were analyzed using multivariate statistics. Results: TOP1 alterations were observed in 4 cell line metaphases. In 3 cases, TOP1 gain involved CEN20, indicating either gain of chromosome 20 or 20q. In all cell lines CEN2 was found to reflect chromosomal ploidy levels and therefore the TOP1/CEN2 probe combination was selected to identify TOP1 gene gains (TOP1/CEN2 ratio ≥ 1.5). A total of 151 patient tumor sections were analyzed with TOP1/CEN2. Among these, 99 patients (65.6%) had TOP1 gain of which 15 patients (10 %) harbored TOP1 amplification (TOP1/CEN20 ratio ≥ 2.0). For OS, TOP1 amplification showed a trend towards longer OS and TTR (HR: 0.59; 95% CI: 0.31-1.14; p = 0.11; HR: 0.50; 95% CI: 0.23-1.09; p = 0.08; respectively), whereas TOP1 gain did not. For LR in rectal cancer patients (n=70), TOP1 gains were significantly associated with shorter time to LR (HR: 1.98, 95% CI: 1.02-3.84; p = 0.04), whereas TOP1 amplifications showed a trend towards longer time to LR (HR: 0.40; 95% CI: 0.14-1.11; p = 0.08). Conclusions: TOP1 gene gain occurs frequently in stage III CRC in a mechanism that often includes CEN20. Using CEN2 as a measurement for tumor ploidy levels, we were able to discriminate between gains and amplifications. TOP1 amplifications showed a trend towards longer OS, TTR and time to LR, whereas TOP1 gains were associated with shorter time to LR. Future studies will investigate the difference between TOP1 gains and amplifications in a larger TOP1 inhibitor treated patient population.
Cell-free EBV DNA in Hodgkin lymphoma.
Authors: Kanakry Jennifer Ann , Gellert Lan L. , Kasamon Yvette L. , Lemas M. Victor , Toure Marie Valerie , Ambinder Richard F.
Published: February 2017

7

Background: EBV is associated with a subset of Hodgkin lymphoma (HL), identified by tissue-based techniques such as EBER in situ hybridization of tumor specimens. Recent evidence suggests that cell-free EBV DNA may be a surrogate for EBER and serve as a tumor marker in HL. Methods: EBV DNA copy number was quantitated by RT-PCR of plasma, serum, and peripheral blood mononuclear cells (PBMCs) from HL patients and compared using Pearson’s correlation. Receiver operator characteristic (ROC) curves determined thresholds for EBV DNA copy number to discriminate EBER status. Kaplan-Meier curves and Cox proportional hazards models were constructed to analyze failure-free survival (FFS). Results: Plasma and serum EBV DNA (n=30 paired samples) were correlated (r=0.96, p<0.0001), while plasma and PBMC EBV DNA were not correlated (n= 25 paired samples, r=0.35, p=N.S.). Detectable plasma EBV DNA was 85% concordant with EBER status (n=34 paired samples) while detectable PBMC EBV DNA had a low concordance of 50% with EBER (n=78 paired samples). In a prospective clinical trial of HL patients treated with rituximab-ABVD, we confirmed the association between pre-treatment plasma EBV DNA and EBER (n=33 paired samples) and found a cut-off of > 60 viral copies/100 µL plasma to have optimal test characteristics with 100% sensitivity, 93% specificity, and 94% concordance with EBER. We observed that EBV DNA was cleared from the plasma of patients with EBER(+) HL within one month of treatment, corresponding to a durable remission in each instance. In a larger prospective trial of HL (E2496), pre-treatment plasma EBV DNA was 95% concordant with EBER (n=121 paired samples), with a sensitivity of 92% and specificity of 96% at the same cut-off of 60 viral copies/100 µL plasma. Pre-treatment plasma EBV DNA positivity was found to be an independent predictor of inferior FFS with a hazard ratio of 2.0 (95% CI 1.2 – 3.5), as presented at ASCO 2012 (abstract #8003). Failure to clear the plasma of EBV with treatment corresponded to inferior 3-year FFS (44% vs 69%, log-rank p=0.03). Conclusions: These findings suggest that cell-free EBV DNA may be a useful tumor marker in HL, demonstrating concordance with EBER, prognostic significance prior to therapy, and utility as a marker of treatment response during therapy.
Trastuzumab-reactive antibodies (TR-abs) in serum and trastuzumab (Tzb) benefit prediction in patients with HER2-overexpressing breast cancer.
Authors: Pohlmann Paula Raffin , Miller Todd W. , Blum David L , Pareh Dipti , Yan Heping , Sutton Cammie R , Dugger Teresa C , Salazar Lupe G , Wu William , Reyzer Michelle L. , Disis Mary L. , Mernaugh Ray , Arteaga Carlos L.
Published: February 2017

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Background: Trastuzumab (Tzb) is a humanized monoclonal antibody (MAb) approved for treatment of HER2-overexpressing breast cancer. Unfortunately not all patients benefit from it and lack of response cannot be predicted. We detected TR-abs in serum of mice and patients treated with Tzb. We hypothesized that TR-abs would associate with response to therapy. Methods: Direct/competition ELISA, dot blot, and mass spectrometry were used to detect and characterize TR-abs in sera from Tzb treated FVBMMTV/HER2transgenic mice, from hybridoma MAbs stemming from transgenic mice responding favorably to Tzb (992-18 mMAb), and in sera of 22 patients with metastatic breast cancer enrolled in a phase I clinical trial. WST-1 viability assay was used to assess biological activity of 992-18 on SKBR3 or BT474 human breast cancer cell lines. Results: From 12 mice bearing HER2-overexpressing tumors and treated with Tzb, 5 responded to therapy and 7 exhibited progressive disease (PD). All 5 responders had elevated TR-abs, whereas TR-abs were low/undetectable with PD (p=0.002; Mann-Whitney two-tailed). This was confirmed in a second cohort of 16 mice, in which TR-abs were undetectable prior to treatment, but gradually detected with Tzb therapy and tumor regression. TR-MAb 992-18 directly targeted also SKBR3 and BT474 in cell-based ELISAs and immunofluorescence assays. Treatment with 992-18 reduced BT474 and SKBR3 cell viability in comparison to isotype-matched control Ab (p<0.0001). In sera from patients with metastatic breast cancer, higher concentrations of TR-abs were significantly associated with lower risk of disease progression (p=0.023, Cox regression, univariate analysis). Conclusions: Low serum TR-abs are associated with poor response to Tzb in mice and with shorter progression free survival in women with HER2 overexpressing stage IV breast cancer. In addition, TR-abs (e.g. 992-18) produced in response to therapy may be pharmacologically active. Results support prospective evaluation of patients undergoing treatment with therapeutic antibodies to determine if this non-invasive immunoassay detecting anti-therapeutic antibodies would predict benefit to therapy.
Interleukin-8 (IL8) and transforming growth-factor beta (TGF-β) as drugable biomarkers of response, progression-free survival (PFS), and overall survival (OS) with pazopanib (PZP): A phase II study in relapsed urothelial cancer (UC).
Authors: Necchi Andrea , Zaffaroni Nadia , Mariani Luigi , Giannatempo Patrizia , Nicolai Nicola , Raggi Daniele , Pennati Marzia , Morosi Carlo , Lanocita Rodolfo , Crippa Flavio , Daidone Maria Grazia , Gianni Alessandro M. , De Braud Filippo G. , Salvioni Roberto
Published: February 2017

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Background: Final results of INT70/09 trial of PZP in 41 multi-relapsed patients (pts) with UC reported one of the highest response-rates as 7 pts (17%) had a confirmed PR, 14 a confirmed SD (51% clinical benefit) after independent review (AACR and ASCO 2012). Final achievements on circulating biomarkers are presented. Methods: From 02/2010 to 07/2011, pts received PZP 800 mg once daily until PD or unacceptable toxicity. 50 mL of EDTA blood samples were collected at baseline (T0) and after 4wks (T1) together with disease restaging in all pts to analyze plasma levels of VEGF, sVEGFR-1,-2 and -3, c-Kit, HGF, TGF-β, IL-6, 8 and 12 by multiplex ELISA plates. Changes in marker levels were analysed with the Wilcoxon signed-rank test and matched with RECIST response with the covariance analysis. A logistic regression and Cox regression model evaluated the association with response probability, PFS and OS. Biomarker level was modelled as a continuous time varying covariate. Results: Significant increase from T0 to T1 was observed for VEGF (p<0.0001), HGF (p=0.017), IL6 (p<0.0129) and IL8 (p<0.0013) and decrease for VEGFR2 and c-Kit (p<0.0001 each). Increasing IL8T1 level associated with lower response probability at covariance analysis (p=0.0104). Both TGF-βT0 (p=0.0019) and TGF-βT1 (p=0.0017) levels associated with PFS while elevated IL8T0 (p=0.0170), IL8T1 (p=0.0107) as well as TGF-βT0 (p=0.0472) and TGF-βT1 (p=0.0013) levels associated with OS. Elevated VEGFT1 also associated with shorter OS (p=0.0084) but significance was lost when jointly modelling biomarkers. Pts having IL8T1 levels < 80 pg/ml had greater response (80%) and 6-month OS (60%) probability than those with a level ≥ 80, for whom a dramatic fall was observed. Conclusions: Both IL8 and TGF-β had a prognostic value while IL8 modulation was also associated with response probability. These markers may serve to select pts most likely to get a benefit from PZP. There is a rationale for a sequence/combination with PZP and a phase II study of an anti-TGF-β receptor ALK1 moAb (PF-03446962) for refractory UC is currently recruiting (NCT01620970).
Comparison of SearchLight and Luminex multiplex assays: A case study of 27 cytokines in 156 melanoma blood serum samples.
Authors: Lin Yan , Butterfield Lisa , LaFramboise William A , Potter Doug M , Lin Hui-Min , Sander Cindy , Kirkwood John M. , Tarhini Ahmad A.
Published: February 2017

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Background: Searchlight and Luminex are two popular multiplex immunoassay platforms. Yet, very few studies have directly compared the performance of these two platforms on real clinical samples. Here we report a case study of 27 cytokines assayed by both platforms on 156 previously banked melanoma serum samples. Methods: Searchlight (Aushon) and Luminex (Invitrogene) assays were carried out according to protocols specified by the manufactures. Intra-experimental coefficients of variation (CV) of the intensities were calculated using the technical duplicates. The detection rate for each marker was calculated based on the cutoffs specified by the manufactures. Spearman’s correlation coefficient (r) was used to quantify the agreement of the measurements of each marker between the two platforms. Wilcoxon signed-rank tests were used to compare CVs and detection rates between the platforms. Results: We observed significantly higher intra-experimental CVs in the Searchlight data (median=8.9%, range=4.9%-11.9%) than in the Luminex data (median=4.1%, range=2%-7.5%, p<0.0001). The CVs were not correlated between platforms (r=-0.08, p=0.69), suggesting that the CVs were not sample specific. We also observed a higher detection rate using the Searchlight platform (median=99%, range=26%-100%) than the Luminex platform (median=94%, range=18%-100%). However, the difference was not statistically significant (p=0.62). Spearman’s r for cytokine concentrations between platforms ranged from -0.13 to 0.86 (mean=0.44). The disagreement for 5 of the 6 cytokines with r<=0.1 can be explained by their standard curves. Conclusions: In this case study of 156 samples, Luminex platform demonstrated significantly better reproducibility measured by CV and the Searchlight platform appeared to be more sensitive measured by the detection rate and the overall range of the measurements. The agreement between the two platforms ranges widely among the markers. Careful examination of the standard curves is critical to interpretation of the cytokine data. Assays of the cytokines by single analyte ELISA is ongoing.
Identification of multifunctional cytotoxic T-cell subsets as immune correlates with clinical outcomes in a phase II study of AGS-003, an autologous dendritic cell-based therapy administered to patients with newly diagnosed, metastatic RCC.
Authors: DeBenedette Mark , Jurisica Igor , Gamble Alicia H. , Tcherepanova Irina Y. , Williams W. Lee , Plessinger Doug , Miesowicz Fred , Nicolette Charles A.
Published: February 2017

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Background: AGS-003 is an autologous dendritic cell (DC) immunotherapy prepared from matured monocyte-derived DC co-electroporated with the subject’s own amplified tumor RNA and synthetic CD40L RNA. The mechanism of action (MOA) of AGS-003 was evaluated in combination with sunitinib for treatment of advanced renal cell carcinoma (RCC) in AGS-003-006, an open label phase II trial including subjects with newly diagnosed, unfavorable-risk, metastatic clear cell RCC. The goal of the immune monitoring platform is to identify unique cytotoxic T-cell (CTL) signatures that correlate with clinical outcome in subjects receiving AGS-003 in combination with sunitinib. Methods: Multiparametric flow cytometry was used to identify tumor-reactive CTL subsets induced by AGS-003 based on combinatorial expression patterns of surface markers CD28, CD45RA, CD27 and CCR7. Moreover, further partitioning of each CTL subset identified combinatorial expression patterns of Markers of Immune Function (MIFs) defined as cytokines (IFN-γ TNF-α, IL-2), cytolytic markers (Granzyme b, CD107) and proliferation. Correlates of CTL signatures with clinical outcome were analyzed using an adaptation of a binary tree-structured vector quantization (BTSVQ) approach, originally developed to cluster and visualize large microarray data sets. The BTSVQ approach implements a two-way unsupervised clustering that allows a subject’s CTL signature to be mapped based on both surface marker and MIFs expression patterns to identify unique clustering patterns linked to clinical outcome. Results: Data analysis identified a unique CTL signature (CD28+/CCR7+/CD45RA- phenotype) displaying a broad MIFs profile as a statistically significant correlate to PFS and OS in patients treated with AGS-003. Conclusions: These results support the intended MOA of AGS-003 in vivo, as the induction of anti-tumor central and effector memory CTL responses. These data warrant further immunological evaluation of AGS-003 in the randomized phase III ADAPT study using AGS-003 in combination with standard treatment in RCC subjects.
Predictive value of preoperative peripheral lymphocyte count on recurrence of resectable non-small cell lung cancer.
Authors: Zhang Jian , Huang Shaohong , Li Hui , Chen Xiuling , Li Yun , Wang Cuiping , Gu Lijia
Published: February 2017

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Background: Recently, the prognostic value of cancer-related inflammatory response has been revealed. Previous studies showed that peripheral neutrophils and lymphocytes had significant impact on the prognosis of advanced and early node-negative non-small cell lung cancer (NSCLC). The purpose of this study was to investigate the prognostic value of preoperative lymphocyte and neutrophil counts in patients with NSCLC who underwent radical surgery and adjuvant chemotherapy. Methods: Retrospective analyses were performed to examine the impact of preoperative peripheral lymphocyte and neutrophil counts on disease-free survival (DFS) and overall survival (OS) and to analyze the relationships of these factors to clinicopathological factors. Results: 142 patients with NSCLC were evaluated totally. 57 (40.1%) patients had local recurrence or metastasis. Multivariate analysesrevealed that peripheral lymphocyte count was an independent favorable prognostic factor of DFS (hazard ratio: 0.548; 95% confidence interval: 0.351-0.857; p=0.008) but not OS (p=0.164). The maximum log-rank statistical value was 9.504 (p=0.002) when the cutoff value of lymphocyte was 1800mm-3. The median DFS were 318.0 days (95% confidence interval: 226.0-410.0) for lymphocyte≤1800mm-3 group and 669.0 days (95% confidence interval: 0.0-1431.0) for lymphocyte>1800mm-3 group. Low lymphocyte count was related with lymphatic invasion (p=0.012) and recurrence of NSCLC (p=0.022). Peripheral neutrophil count had no impact on DFS or OS when analysis included all the 142 patients. In addition, T stage (p=0.001), N stage (p=0.001) and vascular invasion (p=0.021) were also independent unfavorable prognostic factors of DFS. Pathological stage (p=0.001), vascular invasion (p=0.011), and ECOG score (p=0.009) were independent unfavorable prognostic factor of OS. Conclusions: Preoperative peripheral lymphocyte count, which is related with lymphatic invasion, is an independent favorable prognostic factor of DFS in patients with NSCLC who underwent radical surgery and adjuvant chemotherapy.