Internal Negative Control Research Articles

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 °C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 °C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Abstract P1-03-04: Role of PTEN and BRCA1 as determinants of synergy for the combination of vistusertib with carboplatin and olaparib in TNBC

Abstract Introduction: Platinum agents are being used in combination with targeted agents in advanced triple-negative breast cancer (TNBC) (See K. Gelmon et al., 2012). Additionally inhibition of PARP is also being considered as a “targeted” therapy for TNBC (Anders CK et al., 2010). PARP inhibitor (i), Lynparza (olaparib,AstraZeneca)met the primary endpoint of a Phase III trial in which Lynparza was compared to physician's choice of a standard of care chemotherapy in patients with HER2-negative metastatic BC harboring germline BRCA1/2 mutations (BRCAm). Based on cBioportal data analyses and experimental studies we and others have reported that more than 30% PTEN loss in TNBC leads to activation/upregulation of the PI3K pathway (Nature. 2012; Ellis and Perou, 2013; Dey et al., 2012; De et al., 2016; Reed and Shokat 2017;). In line with active kinase profiling, genetic and pharmacological data which defined mTOR as an important target in TNBC (Montero JC et al., 2012), we have demonstrated that mTORi has anti-tumor activity in TNBC (De et al., 2014). Aim: These studies focused on exploring the synergy of biology-based targeted drugs PARPi (olaparib, O), mTOR kinase (vistusertib, V), and platinum (C) in TNBC models. Method: TNBC cells of multiple genetic backgrounds were used to test the combination(s) on proliferation and apoptosis by monitoring growth and using real time Annexin V reagent in a microscopy-based assay (Essen IncuCyte Zoom). Flow cytometric analysis of cell cycle progression by PI staining was also used. Long term clonogenic 3D growth was monitored in matrigel. Results: In BRCAm/PTEN null Sum149 and HCC1937 cells and BRCA wild type (wt)/PTEN null MDA-MB-468 the addition of (V) to (O) and (C) enhanced apoptosis induction and further slowed growth. In Sum149 cells, single agent V treatment induced G1 arrest while O plus C or the triple combination increased S phase accumulation. In MDA-MB-468 cells G1 arrest was seen with V alone and in the triplet. In BRCA wt/PTEN null HCC70 cells V decreased cell proliferation and induced G1 arrest. In the HCC70 model, the addition of O plus C did not synergize with V. In BT20, a BRCA wt/PTEN wt but PI3KCA mutant cell line, no effect on proliferation or apoptosis was seen in the O plus C treated arms. V slowed cell proliferation and increased G1 arrest in a dose-dependent manner. As expected in a BRCA wt/PTEN wt, but RAS active mutant cell line MDA-MB-231 this treatment combination was not effective and was used as an internal negative control. Based on ratios of the normalized slopes of proliferation curves (for triplet), the cells were graded in terms of synergy as SUM149>MDA-MB-468>HCC1937 and HCC70>BT20>MDA-MB231. Treatment with the triplet had the largest effect on reducing 3D colony formation and size as compared to control over single or double treatment. Summary: Here, we present the effect of the combination of vistusertib with olaparib plus carboplatin in several TNBC models. Our data demonstrate that increased effectiveness of the triple combination is seen in cells harboring BRCA1 and PTEN-null mutations. The mechanistic role of these two targets on determining this synergy is being worked out and will be presented at the meeting. Citation Format: Carlson JH, O'Connor MJ, De P, Dey N, Leyland-Jones B. Role of PTEN and BRCA1 as determinants of synergy for the combination of vistusertib with carboplatin and olaparib in TNBC [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-03-04.

Abstract 3411: Rare BRCA2 K3326X increases susceptibility to sporadic pancreatic ductal adenocarcinoma: a PANDoRA study

Abstract Background: The incredibly poor outlook of pancreatic cancer patients underscores an urgent need for early diagnostic markers. Pancreatic cancer ranks third most-frequent among BRCA1/2-deficient cancers with germline mutations detected in <10% of sporadic cases. Germline variants in breast cancer tumor suppressor BRCA2 have been reported to increase predisposition to several cancers including pancreatic tumors. Rare K3326X (rs11571833, c.9976A>T) which introduces a premature stop codon thus truncating the protein, has previously been implicated in familial PDAC, but not in sporadic cases. A frameshift pathogenic mutation c.6503delTT (rs11571658, p.Leu2092Profs) reported to occur in tandem with K3326X in breast and ovarian cancer families, has also been speculated to influence risk associations due to linkage disequilibrium between both variants. Method and results: K3326X was genotyped in 2,969 sporadic cases and 4,700 controls using Taqman chemistry and fidelity of genotypes assessed based on concordance of internal replicates and negative controls. K3326X was observed in 1.2% of cases and 0.8% of controls. Odds ratios (ORs) and associated 95% confidence intervals (CIs) were estimated by multivariate unconditional logistic regression with adjustment for age, sex and region of origin. We also performed a stratified analysis based on age at diagnosis to estimate the risk association between K3326X and early-onset pancreatic cancer. To rule out the likely shared effect of the c.6503delTT mutation on sporadic PDAC risk, we also sequenced DNA from carriers of K3326X in this study. We found K3326X to be associated with increased risk of developing sporadic PDAC ((OR = 1.71, 95% CI = 1.18 - 2.49), P = 0.005). This risk was considerably higher among cases aged 50 years and younger (OR = 2.13, 95% CI = 1.10 - 4.11, P = 0.03). Furthermore, carriers of K3326X did not bear the c.6503delTT mutation thus confirming that the observed risk effect was not influenced by the latter. Conclusion: These robust associations implicate K3326X in the etiology of sporadic and early-onset PDAC, and therefore warrant replication as well as functional studies to elucidate the role of K3326X in DNA repair mechanisms. Citation Format: Ofure M. Obazee, Gabriele Capurso, Angelo Andriulli, Pavel Soucek, Ewa Małecka- Panas, Juozas Kupcinskas, Rudolf Kaaks, Maria Gazouli, Thilo Hackert, Aldo Scarpa, Giulia M. Cavestro, Claudio Pasquali, Hermann Brenner, Daniele Campa, Raffaele Pezzilli, Andrea Mambrini, Beatrice Mohelnikova- Duchonova, Ugo Boggi, Jakob Izbicki, Pavel Vodicka, Elzbieta Iskierka-Jazdzewska, Federico Canzian. Rare BRCA2 K3326X increases susceptibility to sporadic pancreatic ductal adenocarcinoma: a PANDoRA study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3411. doi:10.1158/1538-7445.AM2017-3411

P155 Challenges identifying antibodies using solid phase methods when controls fail

Antibody screening with Solid Phase testing has become mandatory under new UNOS regulations. Single Antigen Beads help discriminate unacceptable mismatches. Technical complications occur when a patient’s sera have a high background level, because potential positive antibodies may be blocked. This is check for using an internal negative control bead (NC). OLI products state, “⋯the NC value should always be lower than 1500 MFI and less than or equal to half of the PC value Vendors agree that treatment using microparticles treated with blocking solution may reduce the background to an acceptable level. However, treatment may be ineffective with some sera, depending on the reason for its’ high background. It is these cases that challenge us to develop additional protocols for assigning antibody s̶pecificity. In an effort to alleviate the majority of these control issues, the current protocol for our lab is to DTT all samples for solid phase antibody testing. However, a certain percentage of these still require further manipulation. Case 1: A Highly Sensitized 49-year-old, Male, Renal Transplant Candidate being evaluated and listed in the UNOS database. After DTT and adsorbing the sera, the NC value was still >5000 MFI. The sera was then diluted @ 1:16 and a NC value of 635 was achieved. At this point the results from DTT, Adsorb and dilution was all correlated. Case 2: A Highly Sensitized 60-year-old, Male, Renal Transplant Candidate being evaluated and listed in the UNOS database. After DTT and adsorbing the sera, the NC value was still >1500 MFI. The sera was then diluted @ 1:16 and a NC value of 206 was achieved. In lieu of performing double or super adsorbing, only to have the NC value still out of range it may be more advantageous to set up a dilution, thus saving time and cost. Results can then be correlating using epitopes and patterns. Performing a dilution can be another tool to use for antibody identification in sera exhibiting a high background.Download : Download high-res image (180KB)Download : Download full-size image

Prognostic Significance of Immunophenotypic Composition of B Cell Acute Lymphoblastic Leukemia at Diagnosis: A Novel Immunophenotype Based Risk Score

Background: Cytogenetic and molecular markers, in addition to age and WBC count, at presentation have been the conventional standards for risk stratifying patients with ALL at diagnosis. There is increased recognition of the oligo-clonal nature of most acute leukemias and hence the limitation of molecular techniques at diagnosis in risk stratification.We hypothesized that the immunophenotype (IPT) heterogeneity as detected by multiparameter flow cytometry (MPFC) will be a function of the complex underlying genetic abnormality and better prognosticate B-ALL at diagnosis.Methods: Retrospectively, the "raw" list mode files of 150 pediatric (0-20years) and 60 adult (>20 years) patients with B-ALL were analyzed (Jan 2009 - Dec 2013). All patients were diagnosed and classified as per WHO 2008 criteria. On MPFC, four blast sub-populations were identified using CD34 and CD45 expression. CD45 expression was divided into bright and dim, using threshold set at one log below CD45 median fluorescence intensity (MFI) of residual lymphocytes while CD34+ population was identified using unstained cells and known internal negative controls (lymphocytes). Four sub-populations were identified and named based upon resemblance to stages of hematogones - Stage 1: CD34+CD45dim, Stage 2: CD34+CD45bright, Stage 3: CD34-CD45bright and stage 0: CD34-CD45dim (Illustrated in Fig 1A). Conventional chemotherapy schedule as previously reported by us was used to treat the patients (Bajel et al. Leukemia:2007).Results: As a continuous variable, proportion of blasts in stage 1, 2, 3, but not stage 0, correlated with events (relapse or death). A high proportion of blasts in Stage 1 had positive impact while high proportion of Stage 2 and 3 had negative impact on EFS. Using uni-variate Cox regression analysis, we tested different cut-offs for all three stages and each sub-population was scored proportional to the hazard ratio as follows: Stage 1 ≤50% = score 2.5, Stage 2 ≥25%: score 2.5 and Stage 3 ≥20%: score 2. All cases were then scored for each parameter and a combined immunophenotype score (IPTS) was calculated. Each patient could have a score varying from 0 to 7. After clustering the KM analysis curves, patients were classified as good risk if the IPTS was 0, high risk if IPTS was >4.5 and the rest were intermediate IPTS. There were significant differences in the clinico-genetic and survival profiles of three categories which are summarized in Table 1 and Figure 1B. On multivariate Cox regression analysis for occurrence of event (including age, high WBC count, BCR-ABL1, cytogenetic risk stratification, BFM-95 risk stratification and post-induction status) IPTS poor risk emerged as an independent prognostic variable (HR:4.7, 95%CI: 1.744-12.697;p=0.002). In adult patients (n=60) similar trends were seen, although, differences were not statistically significant due to small number of patients in which it was evaluated (Figure 1D).Conclusion: Patients with different immunophenotypic composition at diagnosis are associated with distinct clinical profile, genetic features and treatment outcomes. Alone or in combination with a limited conventional molecular profile, this could serve as a low cost but effective strategy to risk stratify patients at diagnosis, especially in a resource limited setting.Table 1Comparison of clinical and genetics features of IPTS risk groups in pediatric cohort.Good(n=65)N (%),Mean ±SDIntermediate(n=63)N (%),Mean ±SDPoor(n=22)N (%),Mean ±SDP valueAge (years)6.0 ±4.211.1 ±5.89 ±6.50.000WBC count (x109/L)11.5 ±15.237.6 ±76.445.8 ±114.70.045BCR-ABL10(0)4(6.3)3(13.6)0.023ETV6-RUNX110(15.4)5(7.9)1(4.5)0.246TCF3-PBX10(0.0)7(11.1)0(0.0)0.018Hyperdiploidy39(60.0)13(20.63)5(22.7)0.000NCI risk stratification0.000Standard53(81.5)22(34.9)10(45.4)High12(18.5)41(65.1)12(54.5)Event9(13.8)20(31.7)13(59.1)0.000Relapse9(13.8)12(19.05)12(54.5)0.000Death7(10.8)17(27.0)9(40.9)0.006 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ENmix: a novel background correction method for Illumina HumanMethylation450 BeadChip.

The Illumina HumanMethylation450 BeadChip is increasingly utilized in epigenome-wide association studies, however, this array-based measurement of DNA methylation is subject to measurement variation. Appropriate data preprocessing to remove background noise is important for detecting the small changes that may be associated with disease. We developed a novel background correction method, ENmix, that uses a mixture of exponential and truncated normal distributions to flexibly model signal intensity and uses a truncated normal distribution to model background noise. Depending on data availability, we employ three approaches to estimate background normal distribution parameters using (i) internal chip negative controls, (ii) out-of-band Infinium I probe intensities or (iii) combined methylated and unmethylated intensities. We evaluate ENmix against other available methods for both reproducibility among duplicate samples and accuracy of methylation measurement among laboratory control samples. ENmix out-performed other background correction methods for both these measures and substantially reduced the probe-design type bias between Infinium I and II probes. In reanalysis of existing EWAS data we show that ENmix can identify additional CpGs, and results in smaller P-value estimates for previously-validated CpGs. We incorporated the method into R package ENmix, which is freely available from Bioconductor website.

P10.20 Digital droplet pcr (ddpcr) quantification of human t-lymphotropic virus type-1 clade c in peripheral blood and lung infiltrates of indigenous australian bronchiectasis patients

Background Human T-cell Lymphotropic Virus type-1 (HTLV-1) infects an estimated 20 million people worldwide causing a fatal T- cell leukaemia and inflammatory conditions, such as HTLV-1-associated myelopathy, resulting from a dysregulated inflammatory response to the virus. HTLV-1 is highly endemic to remote indigenous communities in Central Australia where infection contributes to racial disparities in morbidity and mortality. HTLV-1 related complications are associated with elevated numbers of T-cells in which the HTLV-1 provirus integrates (HTLV-1 proviral load, PVL). Digital-droplet PCR (ddPCR) is capable of absolute quantification of PVL, offering low variability between assays with few cell numbers. We measured HTLV-1 PVLs from buffy coat cells (BCCs) of infected individuals, and bronchoalveolar lavage (BALs) infiltrates from a bronchiectasis patient to determine the range of HTLV-1 copies/cell relative to illness. Methods All samples were from Alice Springs Hospital HTLV-1 cohort (8 BCCs, 1 BAL) and obtained following ethics approval and patient consent in first language. Genomic DNA was extracted from BCCs for ddPCR PVL quantification. HTLV-1 gag/tax primers and FAM/MGB probes were optimised using temperature gradient amplification. Samples were tested in duplicate relative to an internal reference gene standard, RPP30, or negative threshold controls that used VIC/MGB probe. QuantaSoft software was used for data analysis. Results Of the 8 samples from BCCs, the PVL fell between 320 – 8,040 proviral copies per 10 6 cells. The BAL sample PVL was 1,110 DNA copies per 10 6 cells. The sensitivity (or limit of detection) of our ddPCR assay is 2 copies of proviral DNA per 10 4 cells. Conclusion The ddPCR assay demonstrates extremely high sensitivity, low assay-variability and the capability to reliably quantify HTLV-1 PVL. This technique offers logistic advantages in studying relationships between PVL in HTLV-1 patient’s BCCs and BAL. Disclosure of interest statement No Conflicts of interest. ISSTDR and IUSTI recognise the considerable contribution that industry partners make to professional and research activities. We also recognise the need for transparency of disclosure of potential conflicts of interest by acknowledging these relationships in publications and presentations. No commercial contributions have been received for this research project.

HLA Antibody testing- it’s not a one stop assay

HLA laboratories rely on single antigen (SA) assays to determine unacceptable antigens and perform virtual crossmatches (vXM) that can immunologically guide clinicians in the identification of an appropriate donor. Although solid phase antibody detection assays are sensitive, specific and robust-instances arise when further testing is needed to verify results or resolve inconsistencies between these tests. Patient S.T. is a 50 year old male diagnosed with heart failure. Routine work-up in our laboratory is to evaluate % FlowPRA and compare those results to solid phase assays on a Luminex platform which measures the breadth and strength of antibodies in a patient’s sera. Patient S.T. had two samples tested three months apart with a class I FlowPRA of 58% and 62%, respectively. However, The Luminex SA results consistently demonstrated only 5 class I (all B15) antibodies in the positive MFI range leading to a cPRA of about 15%. Moreover, the internal negative control values were > 2500 MFI leading us to believe that there may be additional specificities masked by the high background. How should we deal with these contradictory test results? Can we rely on a vXM to accept a heart for this patient? To reconcile these seemingly contradictory results and to determine if the antibodies detected by Luminex assays were relevant, surrogate FCXM were performed. Surrogate Donor 1 (B15 positive) was FCXM positive. Surrogate donor 2 (B15 negative) was FCXM negative indicating that non-specific binding/false-positivity due to high background should not affect FCXM interpretation, Importantly, testing using phenotype beads were not affected by high background and further confirmed the presence of B15 antibodies. Due to the presence of HLA antibodies and the high background exhibited by this patient, a prospective FCXM must be performed prior to accepting an organ. In conclusion, this is an example in which different tests provide contradictory (or non-consistent) results - and our approach to resolve these ambiguities to support successful organ transplantation.

Abstract 4175: Robust immunohistochemical assay to characterize human cancer tissues for prevalence of vascular endothelial growth factor receptor 3 (VEGFR3)

Abstract VEGFR3 plays a key role in the regulation of lymphangiogenesis in adults, and is also important for tumor angiogenesis and metastasis. In order to characterize human cancer tissues for imunohistochemical (IHC) localization and prevalence of VEGFR3 protein, we developed a robust IHC assay in our lab using a mouse monoclonal primary antibody (Millipore). To optimize the various assay parameters, we utilized high-quality VEGFR3 positive (and internal negative) control tissues (Kaposi's sarcoma, angiosarcoma, lymph node). Reagent negative controls were satisfactory. Specificity of the primary anti-VEGFR3-antibody was supported by a discrete band at the expected molecular weight on western blots using a VEGFR3 transfected cell lysate and negative control cell lines. We also demonstrated lack of specific vascular VEGFR3 staining in pre-absorption experiments with VEGFR3 (but not VEGFR1 or 2) recombinant protein, supporting selectivity of the primary antibody for VEGFR3. Using the fully optimized assay, we stained a human multi-tumor screening tissue microarray (TMA) to demonstrate full range of specific vascular VEGFR3 staining in glioblastoma and carcinomas of the colon, breast, ovary, pancreas, lung, larynx, kidney, cervix, bladder, extrahepatic cholangiocarcinoma and malignant melanoma. Specific, unequivocal VEGFR3 immunoreactivity was interpreted qualitatively (VEGFR3 positive/negative) in tumor blood and lymphatic vessels. No tumor cell staining was seen. Some colon cancer tissues in multi-tumor TMA were VEGFR3+, while others were VEGFR3-. The observed variation in VEGFR3 expression and vascular distribution on screening TMA were further evaluated in two independent cohorts of well-characterized human colorectal cancer tissues (organ-specific TMAs) by a Board-certified, subspecialty GI pathologist (AN), with overall VEGFR3 prevalence rates of 62% (36 of 58 cases) and 56% (55 of 98 cases). Using CD34 and D2-40 IHC assays from a CLIA-certified lab, we confirmed IHC localization of VEGFR3 protein both in the stromal blood vessels and lymphatics within the invasive colorectal cancer stroma. In conclusion, following well-established IHC assay development and standardization protocols and efficient workflow paradigm, we have used a technically robust IHC assay to characterize routinely processed archival human cancer tissues and have demonstrated specific VEGFR3 expression patterns, tissue localization and prevalence in human colorectal cancer tissues. Based on its performance in our hands, this assay can be used to further evaluate patterns of VEGFR3 expression in areas of tumor angiogenesis in other human cancer tissues. Data-driven hypotheses generated from such investigations will be relevant to corroborate VEGF receptor biology and emerging clinical experience with anti-VEGF/anti-VEGFR therapies. Citation Format: Timothy R. Holzer, Drew M. Nedderman, Aejaz Nasir. Robust immunohistochemical assay to characterize human cancer tissues for prevalence of vascular endothelial growth factor receptor 3 (VEGFR3). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4175. doi:10.1158/1538-7445.AM2015-4175

Abstract P4-01-09: Exploring the intra-patient PIK3CA mutational heterogeneity of circulating tumour cells by massive parallel sequencing in patients with metastatic hormone receptor-positive breast cancer

Abstract Introduction: Circulating tumour cells (CTCs) are a real-time reflection of the ad hoc relevant subpopulation in patients with progressive disease. The study comprises the clinical application of a semi-automated methodology to determine the PIK3CA mutational status at a single cell level. Methods: Using CellSearch and DEPArray, we purified single (n=172) and groups (n=93, ranging 5–120 cells) of CTCs from peripheral blood in 18 patients with metastatic ER+/PR+/HER2- breast cancer, of whom archival primary tumour (PT) material was available. Isolation of WBCs served as internal negative control. Recovered cells were subjected to whole-genome-amplification (WGA). During CTC blood draw, an extra SST tube was filled for isolation of circulating cell-free DNA (cfDNA) from serum as comparator. All samples from different compartments underwent gene mutation analysis via targeted amplification of exons 9 and 20 of the PIK3CA gene and downstream 454 massive parallel sequencing (MPS) on the GS Junior system. Results: WGA of single and group CTC samples had a success rate of 83±22 % and 96±8%, respectively. MPS resulted in an average throughput of 135.996±24.423 high quality reads with a normal-distributed mean fold coverage depth of 1257±703 and 1494±897 reads for exon 9 and 20, respectively. PIK3CA mutations were present at a relatively high frequency in archival PT (20/29 (68,9%)) and showed poor and moderate agreement with cfDNA (n=24; 33,3% disparity; kappa=0,03) and CTCs (n=14; 11,28% disparity; kappa=0,329), respectively. Comparison of CTCs and temporally matched cfDNA samples revealed substantial agreement (n=14; 14,3% disparity; kappa=0,536). A concordant PIK3CA status across all compartments (PT, cfDNA and CTCs) was observed in 11/15 (73,3%) samples. At the used sequencing depth, cfDNA failed to the detect PIK3CA mutations in 2 cases (13,3%), which were present in the respective PT and corresponding CTCs. CTCs failed to detect the mutant PIK3CA gene in one case (6,7%), which was present in PT and cfDNA. Gain of mutation was observed in 2/15 patients (13,3%), with a wild-type PT and mutant cfDNA and CTCs at progression. A wild-type PIK3CA sequence in recovered WBCs of all patients (n=15) indicates a high specificity and tumorigenic nature of the picked up variants. In depth intra-patient analysis of mutant CTCs on a single cell level reveals PIK3CA mutational heterogeneity with the presence of both mutant and wild-type CTCs (average %MT/%WT ratio of 50/50). Additionally, unique double-mutated CTCs were detected in 5/15 (30%) cases as well. Conclusions: We report the frequent occurrence of PIK3CA hotspot mutations in metastatic HR+ breast cancer. Intra-patient mutational heterogeneity was observed in the single CTC samples of the same patient. Thereby, this study provides evidence of both concordance and discordance of the PIK3CA genotype in the intravascular compartment (down to the single cell level) with comparison to the temporally unmatched PT. The study presents the utilization of a liquid biopsy, thereby paving the way towards the application of a more personalized medicine in the management of patients with metastatic cancer. Citation Format: Bram De Laere, Dieter JE Peeters, Salgado Roberto, Vermeulen B Peter, Van Dam A Peter, Dirix Y Luc, Van Laere J Steven. Exploring the intra-patient PIK3CA mutational heterogeneity of circulating tumour cells by massive parallel sequencing in patients with metastatic hormone receptor-positive breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-09.

Kinase gene expression profiling of metastatic tumor tissue to prioritize therapeutic targets in clear cell renal cell carcinoma.

476 Background: Kinase inhibitors are the mainstay of therapy for patients (pts) with metastatic clear cell renal cell carcinoma (ccRCC). Since kinases are actionable and lethal metastatic tumor tissue has not been comprehensively studied, we hypothesized that paired intra-patient kinase gene expression analysis in primary tumor (T), matched normal kidney (N) and metastatic tumor tissue (M) may help identify drivers of metastasis and potential therapeutic targets. Methods: Total mRNA isolated from macrodissected formalin fixed paraffin embedded tissue from T, N, and M from 35 pts (total 105 samples) with ccRCC underwent expression profiling for 519 kinase genes using the nCounter System by Nanostring Technologies. Digital raw counts of mRNA abundance were normalized using internal positive and negative controls as well as 7 housekeeping genes. The mean signals from N, T, and M sites were used to calculate change in gene expression, and a p value by t test <0.05 was considered significant. To identify genes unique to metastasis, kinases with significant expression in the M compared to N or T were selected. Functional analysis was conducted to identify key signaling pathways by using Ingenuity Pathway Analysis. Results: The median age of pts was 56 years and 30 were male. Fifteen pts had metastatic disease at presentation and the remaining developed subsequent metastasis. Clustering analysis for all 105 samples separated normal tissues from tumor or metastasis. The top pathways involved in the kinases overexpressed in M compared to N or T involved Pyridoxal 5'-phosphate Salvage, Salvage Pathways of Pyrimidine Ribonucleotides, NF-kB Signaling, NGF Signaling and Cell Cycle Control of Chromosomal Replication. The top kinases over-expressed in M compared to T include: EPHB2, AURKA, GUCY2C, GSG2, IKBKE, MELK, CSK, CHEK2, CDC7, and MAP3K8. Conclusions: Multiple pathways and kinase genes wereover-expressed in metastatic ccRCC, which suggests a major role for agents targeting metabolic pathways and inhibitors of kinases not currently recognized as therapeutic targets. These data warrant validation and suggest potential drivers of metastasis and actionable therapeutic targets in metastatic ccRCC.

Single-Cell Transcriptomics Identifies Intra-Tumor Heterogeneity in Human Myeloma Cell Lines

Multiple myeloma (MM) is a plasma cell neoplasm with significant complexity and heterogeneity. Proteasome inhibitors (PI) including bortezomib (Velcade/Bz), carfilzomib (Kyprolis/Cz) and Ixazomib are effective chemotherapeutic agents in the treatment of MM, used alone or in combination with other anti-cancer agents. However, in spite of the recent improvements in treatment strategies, MM still remains a difficult disease to cure with median survival rate of around 7 years. In a recently published study, we have shown that the heterogeneity in response to proteasome inhibitor (PI)-based treatment in MM is governed by underlying molecular characteristics of the subclones within tumor population (Stessman et al. 2013). We confirmed the presence of residual resistant sub-population comprising up to 15% of the bulk Bz-sensitive cell population in drug-naïve MM tumors. We hypothesize that this pre-existing resistant sub-population may give rise to emerging resistance in course of treatment with PIs.In the current study, we used single cell transcriptomics analysis to identify tumor subclones within Human Myeloma Cell Lines (HMCLs) based on a 48-gene model of predictive genetic signature for baseline PI response. Automated single-cell capture and cDNA synthesis from cellular RNA were performed using Fluidigm’s C1TM Single-Cell Auto Prep System. The cDNA was then harvested and transferred to BioMark HD System for single-cell targeted high-throughput qPCR-based gene expression analysis of a 48 gene-panel using Fluidigm DELTAgene assays. Our 48-gene model combines our previously published 23 gene expression profiling (GEP) signature that could discriminate between sensitive and resistant responsiveness to Bz, and the Shaughnessy et al prognostic 17-gene GEP model along with control genes, including cell cycle genes, anti-apoptotic genes, proteasome subunit genes, house-keeping genes and internal negative controls. Based on the differential expression of these 48 genes used in the modeling, distinct subclonal populations were then identified using a combination of Fluidigm’s analysis software and the R Statistical analysis package. Further, a principal component analysis (PCA) score plot was generated as a two-dimensional grid to visualize the separate populations associated with resistant profiles. Finally, hierarchical clustering (HC) analysis was used to generate heat maps that group expression patterns associated with response.Our results demonstrated the presence of pre-existing subclones of cells within untreated myeloma cells with a characteristic genetic signature profile distinct from the pre-treatment overall (bulk) profile of myeloma cells. As an additional validation of subclonal architecture, we demonstrated the presence of subclones within HMCLs using multi-color flow cytometry. The results presented will help identify the presence and extent of intra-tumor heterogeneity in MM by single cell transcriptomics and may define residual pre-existing subclones resistant to PI therapies. DisclosuresNo relevant conflicts of interest to declare.

Molecular profiling of single circulating tumor cells with diagnostic intention.

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

63-P: QUALITY CONTROL IN THE HLA LABORATORY: THE EFFECT OF INTER-ASSAY VARIABILITY ON SINGLE ANTIGEN BEAD ASSAYS (SABs)

Luminex®-based SAB testing is often used as a semi-quantitative assay to monitor HLA antibody levels pre- and post-transplant. However, the SAB assay is predominantly a manual and non-standardized method that exhibits a high degree of variability. The aim of this study was to determine whether a normalization algorithm using standard controls could be used to correct inter-assay variation. Over 9 months, MFI values for the internal negative (NC) and positive (PC) control beads tested with negative (NS) and positive (PS) control sera (n=264 SAB batches), were plotted. Batches in which the NC and PC MFIs exceeded ±3SD limits were then identified. Informative patient samples (⩾3 sequential sera over ⩽90 days) from these batches were selected and specificities near the positive threshold (2000 MFI) were compared against previous or subsequent runs within 90 days (sequential sera) where NC and PC MFIs were within 3SD. The inter-assay coefficient of variation (CV) was 46% for the NC and 9% for the PC, clearly demonstrating a proportional error. Among 12 batches that exceeded ±3SD limits, sera from 4 informative patients were examined. A normalization algorithm could not be derived because there was no correlation between changes in specific bead MFIs and changes in the NC or PC MFIs within the same batch. The inter-assay variability of SABs complicates and, perhaps, invalidates using MFI values to monitor a patient’s antibody levels over time or following desensitization therapies. These data reveal that SABs are subject to proportional error more pronounced at low MFI levels, making normalization difficult if not impossible. Unless and until the variability of SABs can be reduced through standardization and automation, antibody levels of sequential sera can only be reliably compared when sera are tested in parallel (intra-assay). This strategy, however, comes with a price, namely increased costs of antibody testing.

A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Immunologic Diagnosis of Active Tuberculosis

Clinically evident active pulmonary or extrapulmonary tuberculosis (TB) does not pose a significant diagnostic challenge, but the diagnosis of subclinical or atypical forms of TB is much more troublesome. More accurate tests that can specifi cally diagnose active TB have been much awaited. Interferon-γ (IFN-γ) release assays (IGRAs) are recently developed tests recommended for the diagnosis of latent TB, together with the tuberculin skin test (TST), which had for a long time been the only diagnostic tool for this condition [1]. Although the original intent of IGRAs was for the diagnosis of latent TB, the tests have recently become attractive as a possible option in the diagnosis of active TB, as well. IGRAs measure the in vitro cellular immune responses to Mycobacterium tuberculosis-specifi c antigens, including early secreted antigenic target 6 (ESAT-6) and culture fi ltrate protein 10 (CFP-10), which are not present in any strain of Mycobacterium bovis BCG as well as in many nontuberculous mycobacteria. In contrast, the TST uses a nonspecifi c whole culture fi ltrate of tubercle bacilli containing over 200 antigens. Th is leads to its low specifi city. Two types of IGRAs are now commercially available. Th e fi rst is QuantiFERON-TB Gold (QFT-G; Cellestis), an assay that uses the patient’s whole blood to measure the level of IFN-γ from the stimulated supernatant. A variant method, QuantiFERON-TB Gold In-Tube (QFT-GIT), uses tubes prefi lled with antigens. The second type of IGRA, T-SPOT.TB (Oxford Immunotec), uses the enzyme-linked immunospot (ELISPOT) assay to measure the number of IFN-γ-secreting T cells on stimulation by M. tuberculosis-specifi c antigens. A fi xed number of peripheral mononuclear cells is used in this assay. Both types of IGRAs have internal positive and negative controls to guard against technical errors. Th e failure of the controls in the tests, defined as indeterminate test results (ITRs), means that the reliability of results cannot be guaranteed. Th e incubation step in IGRAs selectively amplifi es replication of eff ector memory T cells, since central memory T cells require a longer period of in vitro incubation than eff ector memory T cells. Central memory T cells are major components within the indurated skin produced by the TST. Th erefore, IGRAs are more likely to be positive in persons recently infected with M. tuberculosis [2]. Assessing the accuracy of IGRAs in diagnosing M. tuberculosis infection is difficult due to the absence of a gold standard to confirm a diagnosis of latent TB infection or culture-negative active tuberculosis. Th us, the performance of the tests has been measured based on the epidemiology of TB or by using cases of active TB. Studies regarding household contacts with active TB showed that the pooled sensitivity of IGRAs for the prediction of active TB after exposure was 80 to 90%, the specifi city 56 to 83%, the positive predictive value (PPV) 4 to 8%, and the negative predictive value (NPV) 99 to 100%. For the TST, the sensitivity was 90 to 100%, the specifi city 29 to 39%, the PPV 2.7 to 3.1%,

Detection of Neisseria meningitidis DNA in blood samples using direct-PCR test

BackgroundMeningococcal disease caused by Neisseria meningitidis is a widely distributed complex human disease affecting all age categories. Successful and effective treatment of patients with this disease depends on precise and early diagnosis of the disease.ObjectiveThe aim of this study was to evaluate the possible use of direct PCR (D-PCR) for the detection and amplification of N. meningitidis DNA in blood samples compared with the conventional PCR (C-PCR) method, which needs bacterial culture and DNA extraction.Materials and methodsSpecific primers on the basis of the 16S rDNA of N. meningitidis were used for the amplification of 600 bp DNA fragment. Two strategies were followed: D-PCR, which relies on amplification of DNA directly in blood without DNA extraction using the KAPA Blood PCR Kit, and the C-PCR, which relies on the extraction of bacterial DNA using the Qiagen QiAmp DNA Mini Kit. The following blood samples were included in each strategy: A blood sample with bacterial cerebrospinal meningitis confirmed by blood culture, a normal blood sample seeded with N. meningitidis ATCC (13090) reference strain as positive control for the standardization of the PCR procedure, a normal blood sample as a negative control, and an internal negative control test sample (H 2 O).ResultsBoth D-PCR and C-PCR tests gave the expected amplified DNA fragment of 600 bp on agarose gel electrophoresis of both patients' blood sample and N. meningitidis seeded normal blood sample, whereas no amplified products were detected when both tests were performed on normal blood sample or the internal negative H 2 O control.ConclusionDirect blood PCR assay could be a possible easy, rapid, nonexpensive, and specific method for the detection of meningococci in blood samples, particularly in situations in which culture is difficult because of previous treatment, and also could facilitate the large-scale screening of various medical conditions.

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators. To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (http://www.ibridgenetwork.org/wustl/splinter). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (http://www.ibridgenetwork.org/wustl/splinter). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Occurrence of Human Enteric Viruses in Commercial Mussels at Retail Level in Three European Countries

In this study, the prevalence of different enteric viruses in commercial mussels was evaluated at the retail level in three European countries (Finland, Greece and Spain). A total of 153 mussel samples from different origins were analysed for human norovirus (NoV) genogroups I and II, hepatitis A virus (HAV) and hepatitis E virus (HEV). Human adenovirus (HAdV) was also tested as an indicator of human faecal contamination. A full set of controls (such as sample process control, internal amplification controls, and positive and negative controls) were implemented during the process. The use of a sample process control allowed us to calculate the efficiencies of extraction, which ranged from 79 to 0.5 %, with an average value of 10 %. Samples were positive in 41 % of cases, with HAdV being the most prevalent virus detected (36 %), but no significant correlation was found between the presence of HAdV and human NoV, HAV and HEV. The prevalences of human norovirus genogroup II, HEV and human NoV genogroup I were 16, 3 and 0.7 %, respectively, and HAV was not detected. The estimated number of PCR detectable units varied between 24 and 1.4 × 10(3) g(-1) of digestive tract. Interestingly, there appeared to be a significant association between the type of mussel species (M. galloprovincialis) and the positive result of samples, although a complete overlap between country and species examined required this finding to be confirmed including samples of both species from all possible countries of origin.