21st San Diego Conference:
Genomic Technologies at the Interface of Diagnostics and Therapeutics
March 25-26, 2008
Coronado Bay Resort & Spa
Coronado, CA
AACC thanks all of the scientists who shared their work at this conference. Please scroll down to view their efforts.
A molecular-based biosensor configured in a manual/rapid format
Heidi Koss1, David Latorra1, Diane Weed2, Jacinta Schraufnagel1, Joshua Klonoski1, Ron Torres3, Robert Jenison1
1Great Basin Scientific, Longmont, CO; 2Denver Health Hospital, Denver, CO; 3Utah State University, Logan, UT
The incidence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically increased over the past decade. If not diagnosed and treated appropriately, these human pathogens can cause a range of life-threatening illnesses including septicemia and toxic shock syndrome. Early diagnosis and treatment of these infections has been associated with improved outcomes in patients. Clinical outcome studies have shown that reducing the time to diagnosis decreases the patient’s length of stay, leading to significant cost savings to the hospital, and reduces morbidity and mortality. Recently, two relatively rapid molecular-based tests for detecting MRSA have been introduced to the clinical setting. These tests rely upon using a real-time PCR platform requiring expensive instrumentation that may not be available in smaller hospitals or point of care settings. We describe here the development of a novel and inexpensive on-chip amplification/detection platform. The amplification is isothermal at 65 C, requiring only a simple heat block, and the signal is permanent and detectable by the naked eye on simply configured silicon chips. Analytical sensitivity of 10 copies of genomic DNA per sample has been demonstrated. From either a 10 uL blood culture aliquot or a nasal swab, organisms are extracted, amplified and detected within 60 minutes. The test may be done manually, with fewer than 12 total steps and 10 minutes active time, so it is configured appreciably like simple rapid tests used currently to detect primarily respiratory viruses. We recently validated the platform by testing 22 clinical blood culture samples. The described method showed 100% concordance for identification of MRSA with standard microbiological methods that take 24-72 hours to determine a result.
Fabrication of M. tuberculosis whole-proteome microarray chip for serodiagnostic antigen discovery
X. Liang1, D. Molina1, V. Huynh1, S. Kunnath2, H. Wang2, A. Davidow2, G. Michel3, M. Perkins3, P. Felgner4, M. L. Gennaro2.
1ImmPORT Therapeutics, Inc., Irvine, CA; 2Public Health Research Institute, UMDNJ, Newark, NJ; 3Foundation for Innovative New Diagnostics, Geneva, Switzerland; 4University of California-Irvine, Irvine, CA
Developing accurate serodiagnostic assays for active tuberculosis has been difficult because 1) no single antigen is recognized by serum antibodies in every tuberculosis patient, making it necessary to utilize multi-antigen cocktails, and 2) the high prevalence of asymptomatic infection in high-burden regions makes it necessary to identify markers that distinguish active tuberculosis from other stages of Mycobacterium tuberculosis infection as well as from other pulmonary disease. These facts create a tremendous challenge for developing diagnostics: not only is it necessary to identify antigens that do not cross-react with non-TB conditions, but also it is necessary to identify antigens that discriminate between active disease and asymptomatic infection. To overcome this challenge, it is necessary to interrogate a large number of M. tuberculosis antigens and a large number of sera. We have undertaken a novel approach involving serological interrogation of the entire proteome of M. tuberculosis to identify antibody markers characteristic of active tuberculosis. By taking advantage of a newly developed, high-throughput platform for gene cloning, protein expression and microarray fabrication, we have generated a whole-proteome chip for M tuberculosis. The proteome chip was probed with ~1,000 individual sera from patients having active tuberculosis and various control groups (latent infection, past tuberculosis, non-infected subjects, other pulmonary disease). Data were used to explore normalization and classification strategies leading to the identification of a small subset (<50) of antibody markers that classify active TB with high accuracy (>95% specificity and >80% sensitivity).
Greater silencing of KIF11 gene by siRNA in human cancer cell lines using LipofectamineTM RNAiMAX
Liu C, Dalby B, Solbach J
Invitrogen Corporation, Carlsbad, CA
RNAi is widely used for gene function analysis in vitro and therapeutic developments in vivo. The considerable efforts have been made to maximize the silencing efficiency by improving siRNA design, stability in serum, and transfection reagents for sufficiently delivering siRNA into cells. Here, we are using siRNA targeting KIF11, a kinesin-related motor involved in mitotic spindle assembly (also known as Eg5) as an example to access the silencing efficiency for phenotypic changes in several human cancer cell lines. We transfected cell lines such as HeLa, MCF-7, HCT116 and A549 with Lipoplexes of 1-10 nM KIF11 StealthTM Select RNAi – 25 bp long double dsRNA with proprietary modifications on sense strand, and complexed with cationic lipid formulation -LipofectamineTM RNAiMAX. A greater than 90% knockdown of Kif11 gene activity was observed in the most of cell lines at 24 hrs posttransfection. Meantime, the formation of round-shaped cells was observed under phase-contrast microscope. Staining cells with Propidium Iodide (PI) during 24 hrs after transfection showed that round-shaped cells were living cells, suggested that cells phenotypic changes was due to the KIF11 gene silencing in these cells, not the toxicity caused by transfection reagent. Immunofluorescence staining with anti-tubulin antibodies was shown that centrosomes in dividing cells fail to separate and form bipolar spindles, and result in formation of monopolar spindles in cancer cell lines. These results indicate the effective silencing KIF11 gene with RNAi technology induces mitotic arrest and apoptosis in human cancer cells, functions as well as cancer therapeutic drug monastrol or S-trityl-L-cysteine.
Correlation of JAK2 V617F mutant allele quantitation with clinical presentation and type of chronic myeloproliferative disease
Malysz J, Eley T, Meng X, Crisan D
Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, MI
Introduction: An activating JAK2 V617F mutation is postulated as the pathogenetic basis of the disease process in many patients with chronic myeloproliferative diseases (CMPDs). This mutation has been reported in almost all patients with polycythemia vera (PV) and in approximately one half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Our goal was to correlate JAK2 mutant allele quantity with clinical presentation and type of chronic myeloproliferative disease. Methods: We reviewed clinical course and laboratory data of 70 (N=70) retrospectively selected patients with BCR/ABL negative, JAK2 positive CMPDs with JAK2 quantitation performed between February 2006 and December 2007. DNA samples extracted from bone marrow or peripheral blood specimens were amplified using real-time PCR with fluorescent labeled allele specific PCR primers. The amount of JAK2 V617F mutant allele was quantitated using a standard calibration curve. The detection limit of our method was 1% mutant allele. Quantitation results equal to or greater than 50% denote homozygosity of the hematopoietic clone. Patients with results lower then 50% were presumed to be heterozygous, however possible homozygosity of a small clone in these cases cannot be entirely ruled out by our present methodology. We correlated JAK2 quantitation with clinical and laboratory parameters including hemoglobin levels, white blood cell (WBC) and platelet counts, spleen size and/or presence of cardiovascular complications such as thrombo-embolic events, bleeding and myocardial infarction (MI). Results: 56 patients (80%) had <50% mutant allele (presumed heterozygous) and 14 patients (20%) were homozygous for JAK2 V617F mutation. Among homozygous patients PV was diagnosed significantly more often than in heterozygous patients (71% vs. 25%; p=0.003). In addition homozygous patients within the PV group had higher mean WBC count (19+/- 10 vs. 11+/- 4; p=0.043). ET patients were more likely to be heterozygous (48% vs 7%; p=0.005). Splenomegaly or history of splenectomy was more frequent in homozygous patients independent of diagnosis (89% vs.48%; p=0.03). Concomitant cardiovascular complications were recorded only in a minority of patients. Incomplete clinical data did not allow statistical analysis of its occurrence. Cardiovascular complications were reported in 7 of 14 homozygous patients (50%) and 12 of 56 heterozygous patients (21%). Our study revealed the highest quantity of JAK2 mutant allele in PV patients, followed by chronic myeloproliferative disease-unclassifiable (CMPD-U) and ET patients. Low number of IMF patients precluded statistical analysis. Conclusions: Homozygous patients were more frequently diagnosed with PV. ET patients were more frequently heterozygous. Among PV patients, homozygosity was associated with significantly higher mean WBC count. Splenomegaly or history of splenectomy was more frequent in homozygous patients regardless of diagnosis. Quantitative JAK2 analysis appears an important contributor to CMPD evaluation and may aid in stratification of patients into subgroups with different frequency of complications resulting in differences in morbidity and mortality.
Simple integrated process for diagnosis by DNA chip: An example of Papillomavirus detection
Kim KT, Oh SJ, Chae C-B
Institute of Biomedical Science and Technology, Konkuk University, Seoul, Korea
Microarrays of single-stranded oligonucleotides immobilized on solid support are used for many purposes: identification and classification of infectious agents such as viruses, disease-causing mutations of various genes, expression of genes, etc. One of the major advantages of using microarray is the ability to provide information on many gene sequences all at once. However, the technology is cumbersome and difficult to be adopted by hospitals and clinics for routine and high throughput diagnosis. For example, nucleic acids (DNA or RNA) have to be extracted from clinical specimens, target genes need to be amplified, and the amplified nucleic acids need to be hybridized with the capture probes immobilized on the surface of solid support such as glass plates. Thus, the process requires many pipettings and manipulations by highly trained work force and lasts one full day.
We have developed a very simple integrated process which carries out gene amplification and hybridization on the surface of DNA chip coated with dendrons without prior purification of nucleic acids from biological specimens. Such process has been successfully applied for clear detection and identification of different genotypes of human papillomavirus from clinical specimens of human cervical cells with high sensitivity. For example, human cervical cells are briefly heated at 95oC and centrifuged. The supernatant is added to PCR mixture, and combined PCR and hybridization is carried out, without changing solution, on the surface of a dendron-coated glass slide with the immobilized oligonucleotide capture probes. The process with the heated cells produces very similar result as the diagnosis with purified DNA in terms of accuracy, sensitivity, and reproducibility. The whole process takes about 3 hours with minimal requirement of human attention. The integrated process has the following benefits: saving of labor, time, and material costs. It also offers opportunity for automation of the diagnosis by DNA chip.
A multiplex, real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level
Pinsky BA1, Banaei N1,2
1Department of Pathology, Stanford University School of Medicine, Stanford, CA; 2Clinical Microbiology Laboratory, Stanford Hospital and Clinics, Palo Alto, CA
The species identification of members of the Mycobacterium tuberculosis complex is critical to the timely initiation of both appropriate antibiotic therapy and proper public health control measures. However, the current commercially available molecular assays only identify mycobacteria to the complex level and are unable to differentiate M. tuberculosis from the closely related M. bovis and M. bovis BCG. We describe here a rapid and robust, two-step, multiplex, real-time PCR assay based on genomic deletions to definitively identify M. tuberculosis, M. bovis, M. bovis BCG and other members of the complex. When tested against a panel of well-characterized mycobacterial reference strains, the assay was both sensitive and specific, correctly identifying all strains. We applied this assay to 60 clinical isolates previously identified as M. tuberculosis complex and found 57 M. tuberculosis isolates as well as three M. bovis BCG isolates from patients who had received intravesical BCG. Furthermore, analysis of 15 clinical specimens previously identified as M. bovis by spoligotyping revealed an isolate of M. tuberculosis that had been misidentified. We propose that this assay will allow for the routine identification of M. tuberculosis complex members in the clinical laboratory.
Microarray analysis of peripheral lymphocytes before and after fenofibrate treatment in hypertriglyceridemic individuals—the GOLDN study
Cao J1, Tsai MY1, Zhang H2, Nossova N2, Arnett DK3, Hanson NQ1, Liew CC2
1Department of Lab Medicine and Pathology, University of Minnesota, Minneapolis, MN; 2Genenews Inc., Toronto, Ontario; 3Department of Epidemiology, University of Alabama, Birmingham, AL
Background: Fenofibrate is widely used for the management of hyperlipidemia. Its pharmacologically active metabolite fenofibric acid activates peroxisome proliferator-activated receptor-α (PPAR-α) and results in increased lipolysis and plasma clearance of atherogenic triglyceride (TG) rich lipoproteins and reduced TG synthesis. Fenofibrate also has other beneficial effects such as increasing cholesterol efflux and reducing lipoprotein oxidation. PPAR-α independent action of fenofibrate has also been reported. The pharmacological mechanism underlying these effects is not well defined.
Methodology: In the current study, we selected 25 participants with hypertriglyceridemia (baseline plasma TG levels >200 mg/dl) from the Genetics of Lipid Lowering Drugs and Diet Network study, and compared their gene expression profile in peripheral lymphocytes before and after treatment with 3-week open-label fenofibrate (160 mg/d). RNA was extracted within 4 hours of blood collection, and analysis of microarray results was done by Genenews™. Paired t tests were used for comparing before and after treatment.
Results: The mRNA levels of BCL-6 (B cell lymphoma-6) and split ends (Spen) homolog gene in peripheral lymphocytes increased to 1.8 and 1.7 fold respectively after three weeks of fenofibrate treatment. We postulate that the binding of fenofibrate to PPAR-δ/BCL-6 complex releases BCL-6 protein and de-represses the transcription of BCL-6 gene. This action may also activate PPAR-δ and resulting in increased cholesterol efflux. The homolog of Spen in human-SHARP-is a transcriptional co-repressor for PPAR-δ and suppresses the target gene of PPAR-α. The finding that the SHARP gene is upregulated after fenofibrate is evidence for a feedback inhibition mechanism.
Conclusion: The gene expression analysis in peripheral lymphocytes from hypertriglyceredimic participants before and after fenofibrate treatment revealed the dual-activation effect of fenofibrate treatment on PPAR-α and PPAR-δ and confirmed the “gate-keeper” role of PPAR-δ for other PPAR isoforms. Increased understanding of drug mechanism may lead to the better use of combined and /or individualized therapies for raising high density lipoprotein cholesterol (HDL-C) levels.
Determination of genotypes for warfarin sensitivity using PCR directly from whole blood
Naranatt P, Cochran D, Lopez R, Liu Y, Jacobs AR, Reed MR, Coty WA
Osmetech Molecular Diagnostics, Pasadena, CA
Objective: To evaluate the use of a single-step, five-minute whole blood preparation as a sample type for PCR amplification and genotyping of polymorphisms related to warfarin metabolism and sensitivity.
Background: Polymorphisms in the genes for cytochrome P450 2C9 (CYP2C9), vitamin K epoxide reductase complex 1 (VKORC1) and CYP4F2 have been shown to affect warfarin metabolism or sensitivity. Genotyping tests for these polymorphisms can provide information to determine the optimal warfarin dosage; reducing the assay turn-around time between sample collection and result will allow physicians to select the initial dose more rapidly.
Methodology: Peripheral whole blood was collected and used within two weeks of venipuncture. Samples were processed according to the manufacturers’ instructions using QuickExtract™ DNA Extraction Solution (Epicentre Biotechnologies) or Extract-N-Amplify (Sigma Aldrich). Whole blood samples were diluted between 12- to 100-fold with extraction solution, heated at 65°C for two min and then at 98°C for two min (QuickExtract) or incubated at room temperature for 5 min (Extract-N-Amplify). No centrifugation or separation steps were performed. The treated samples were amplified and genotyped using the eSensor® test for CYP2C9 (8 polymorphisms), VKORC1 (-1639G>A) and CYP4F2 (V433M) according to the procedure used for extracted genomic DNA.
Results: Both whole blood preparation methods were compatible with the eSensor® genotyping test and gave a 100% first-pass correct call rate using a 25- to 100-fold (QuickExtract) or a 12- to 21-fold (Extract-N-Amplify) dilution of whole blood. Compared to purified genomic DNA, the yield of individual amplicons from processed whole blood varied from 50% to 134%. In a separate experiment, the effect of potential interfering substances was determined by addition to whole blood of either human serum albumin (3g/dL), bilirubin glucuronide (0.3 mg/mL), human IgG (3g/dL), triglycerides (500 mg/dL), hemoglobin (~20 g/dL) or EDTA (a 5-fold increase in concentration over the standard level of anti-coagulant). All samples gave first-pass genotyping calls with the expected genotype. In a third study, blood samples from 23 random donors gave 100% concordant results between samples processed using the QuickExtract and Extract-N-Amplify methods and a standard genomic DNA extraction method. These 23 samples included the following genotypes: 6 2C9*2 heterozygotes, 2 2C9*3 heterozygotes, 8 VKORC1 heterozygotes and 4 VKORC1 homozygous minor allele.
Conclusion: Rapid methods for processing whole blood samples for PCR and genotyping without separation steps or isolation of genomic DNA are compatible with PCR and genotyping of polymorphisms related to warfarin sensitivity using the eSensor® DNA detection technology. This approach reduced overall time to result by approximately one hour, thus allowing more rapid initiation of pharmacogenetic-based warfarin dosage.
Identification of commonly aberrant genomic regions using high resolution oligo array CGH of FFPE breast cancer samples
Carmack C
Agilent Technologies, San Jose, CA
Several common chromosomal aberrations have been identified in breast cancer. For instance, amplification of the portion of 17q encompassing the ERBB2 oncogene has been estimated to be present in 10%-34% of breast cancer cases. Other aberrations, especially common in estrogen receptor (ER) positive cases include loss of heterozygosity at 16q and gain at 1q. It is important to accurately delineate copy number transitions and boundaries to define the genes that consistently lie within aberrations. Comparative genomic hybridization provides a means to determine the changes in genomic copy number and transition points. We applied high resolution microarray-based (aCGH) analysis of formalin fixed paraffin embedded invasive ductal breast carcinoma samples. We used a 244K CGH array which consists of 244,000 in situ synthesized 60-mer oligonucleotide probes on a single 1 in. x 3 in. slide. This feature density results in an average genomic distance between probes of ~6 kbp across the entire human genome. We analyzed 58 invasive ductal carcinomas, which included 18 erbB2 positive (3+ immuno and/or FISH positive), 7 erbB2 2+ positive (via immunohistochemistry), 24 ER+ and 34 ER negative, 6 Grade I, 25 Grade II, and 27 Grade III tumors derived from archival formalin fixed paraffin embedded (FFPE) tissue from UC Davis. Altered genomic regions were identified using Nexus 2.0 with BioDiscovery’s Rank Segmentation algorithm. Class comparisons, using FDR correct Fisher Exact testing, was utilized on the samples.