Absolute Quantitative PCR Research Articles

Absolute quantitative PCR for detection of molecular biomarkers in melanoma patients: A preliminary report

BackgroundMalignant melanoma is the most malignant tumours of skin and mucous membranes mainly due to its aggressive biological behaviour and tendency to generate early metastases. Unfortunately, the mechanisms underlying the development, progression and the expression of an aggressive melanoma phenotype still remain largely unknown. ObjectivesThe purpose of this study was to determine whether a multi-panel of molecular transcripts can be predictive for risk of recurrent disease in malignant melanoma patients. ResultsPeripheral blood was collected from 31 malignant melanoma patients in follow-up for melanoma and from 30 healthy volunteers randomly selected. Each specimen was examined by qRT-PCR analysis for the expression of six markers: PAX3d, TYR, MITFm, MCAM, TGFβ2 and ABCB5. Malignant melanoma patients expressed an important number of markers, with a median value of four markers. Only PAX3d displayed a trend in terms of differences when the levels of gene expression were made in function of Breslow index. Furthermore, PAX3d showed the best diagnostic capacity among the remaining residual markers or in combination with TGFβ2 and MTIF. ConclusionsWe demonstrated the usefulness of multimarker qRT-PCR to detect circulating melanoma cells in blood and to potentially assessing patient disease status or progression, especially when PAX3d was used in combination with MTIFm and TGFβ2.

Quantitative study of epigenetic signature in head and neck squamous cell carcinoma.

The aberrant upregulation of Forkhead box protein M1 (FOXM1) plays a fundamental role in cancer initiation by perturbing stem cell differentiation. This study aims to investigate the role of FOXMI in epigenetic modification and gene expression of target genes in primary human oral keratinocytes, squamous cell carcinoma cell lines, and head and neck squamous cell carcinoma (HNSCC) tissue biopsies. A genome-wide promoter methylation microarray was used to compare HNSCC cell line (n = 8), primary human oral keratinocytes (NOK; n = 8) transduced with FOXM1 and EGFP, and HNSCC tissue biopsies (n = 3). Seventeen Foxm1B- induced differentially methylated genes were shortlisted. An absolute quantitative polymerase chain reaction was used to validate the differential promoter DNA methylation of each candidate gene induced by FOXML. These results were compared with the methylation status and altered gene expressions of candidate genes in a panel of genomic DNA and messenger RNA (mRNA) samples previously extracted from HNSCC tissue biopsies. The results were consistent with our hypothesis, showing that aberrant. upregulation of FOXM1 expression in in vitro primary NOK induces a global hypomethylation pattern similar to the HNSCC cell line and has an inverse correlation with in vivo mRNA expression levels of HNSCC tissue biopsy. Such epigenetic changes have tremendous clinical potential as biomarkers for early cancer detection and therapeutic interventions.

Absolute qPCR for measuring telomere length in bone marrow samples of plasma cell disorders.

Telomere length (TL) is currently used as an emerging biomarker in understanding the development/progression of hematological malignancies. The absolute quantitative PCR (qPCR) methodology has allowed the study of TL from a variety of mammalian tissues, but it has not been tested for bone marrow (BM) samples. In this study, we have examined the relationship between TL data generated by absolute qPCR versus those obtained by terminal restriction fragments (TRF) in 102 BM samples from patients with plasma cell disorders. A significant linear correlation between both methodologies was observed (p < 0.0001; r (2) = 0.70). Results were also analyzed in relation to clinical characteristics and significant associations between telomere shortening and parameters of adverse prognosis were observed. Furthermore, another set of 47 BM samples from patients with low quantity of DNA for TRF assay were suitably analyzed by qPCR, indicating the usefulness of the absolute qPCR methodology for the inclusion of patients with scarce material to the study. Taken together, these findings are of interest considering the importance of telomere dysfunction in the pathogenesis of cancer and give a new alternative to measure TL in hematologic disorders with substantial time and cost savings.

Selection of suitable endogenous reference genes for relative copy number detection in sugarcane.

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

Variation in Telomere Length of the Common Carp,Cyprinus carpio(Cyprinidae), in Relation to Body Length

Determinations of the age structure of wild teleost populations are vital for sustainable management and conservation efforts. Yet the commonly applied increment-based ageing techniques are limited in some teleost species due to the subjectivity and interpretation of increment patterning, and destructive sampling. Here the application of telomere length as an alternate age determinate was assessed in wild caught specimens of the common carp (Cyprinus carpio). Telomere lengths were measured from muscle biopsies and fin clips using the absolute quantitative PCR method and correlated to specimen fork lengths. Measures of telomere lengths did not differ between sexes or states of sexual maturity. However, tissue type influenced telomere lengths, with telomere lengths measured in muscle biopsies significantly increasing with increased fork lengths of C. carpio. No discernable trend was seen for fin clip derived telomeres. High levels of variability in telomere length within and among size cohorts suggest that ...

Regulation of N-acetyl cysteine on gut redox status and major microbiota in weaned piglets1

This study was conducted to explore the regulation of N-acetyl cysteine (NAC) on gut redox status and proliferation of selected microbiota in weaned piglets. A total of 150 newborn piglets from 15 litters were randomly divided by litter to the control group (normally suckling), the weaning group (fed the basal diet), and the NAC group (basal + NAC diet) with 5 litters per group. Activities of total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and inhibition capacity of hydroxyl radical (IHR), and contents of malondialdehyde (MDA), H2O2, and NO in the ileum, colon, and cecum were analyzed to profile oxidative stress states. The real-time absolute quantitative PCR reaction was employed to quantify the amounts of total bacteria, Lactobacillus, Bifidobacterium, and Escherichia coli. The N-acetyl cysteine, as a universal antioxidant, was used to improve the redox status. Results showed that weaning stress resulted in the occurrence of gut oxidative stress and changes of gut microbiota (P < 0.05). Compared with the weaned piglets, the activities of ileal, colonic, and cecal T-AOC; ileal and colonic GSH-Px; cecal SOD; and colonic and cecal IHR were enhanced (P < 0.05), and the concentrations of ileal and cecal H2O2, ileal and colonic NO, and colonic MDA were reduced (P < 0.05) in the NAC-treated piglets. An increase (P < 0.05) in gut Lactobacillus and Bifidobacterium, accompanied with a decrease (P < 0.05) in Escherichia coli counts, was also observed in the NAC group. Bivariate correlation indicated that Lactobacillus and Bifidobacterium were positively correlated (P < 0.05) with the activities of T-AOC, GSH-Px, and SOD and inversely related (P < 0.05) to increased levels of H2O2, NO, OH, and MDA, and Escherichia coli showed a strong positive association (P < 0.05) with increased levels of free radicals and MDA and a negative association (P < 0.05) with the activities of antioxidant enzymes in intestines of weaned piglets. We concluded that NAC constructively regulated on the changes of the gut redox status and microbiota in piglets in response to weaning stress. The observed correlations implied that the NAC effects on the gut microbiota were confirmed, partly through an effect on oxidative stress in piglets, providing evidence that gut microbiota may be potentially improved by the modulation of the redox status by an antioxidant, which has relevance for gut health and function.

Identification of FOXM1‐induced epigenetic markers for head and neck squamous cell carcinomas

Epigenetic reprogramming of the methylome has been implicated in all stages of cancer evolution. It is now well accepted that cancer cells exploit epigenetic reprogramming, a mechanism that regulates stem/progenitor cell renewal and differentiation, to promote cancer initiation and progression. The oncogene FOXM1 has been implicated in all major forms of human cancer. We have recently shown that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a number of FOXM1-induced epigenetic markers. Differential promoter methylation and gene expression in clinical specimens were measured using commercially available bisulfite conversion kits and absolute quantitative PCR, respectively. Here, we investigated 8 FOXM1-induced differentially methylated genes, SPCS1, FLNA, CHPF, GLT8D1, C6orf136, MGAT1, NDUFA10, and PAFAH1B3, using human head and neck tissue specimens donated by 2 geographically independent patient cohorts from Norway and the United Kingdom. Two genes (GLT8D1 and C6orf136) were found to be differentially expressed in head and neck squamous cell carcinomas (HNSCCs). Using methylation-specific quantitative PCR, we confirmed that the promoters of GLT8D1 and C6orf136 were hypo- and hypermethylated, respectively, in HNSCC tissues. Given that epigenetic change precedes gene expression, methylation status of candidate genes identified from this study may represent a signature of premalignancy, rendering them potentially useful predictive biomarkers for precancer screening and/or therapeutic targets for cancer prevention.

Periodontopathogens levels and clinical response to periodontal therapy in individuals with the interleukin-4 haplotype associated with susceptibility to chronic periodontitis

Periodontitis is an inflammatory disease that results from an interaction between dental biofilm agents and the host immune-inflammatory response. Periodontopathogenic organisms, such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, as well as the host's susceptibility, represented by the host's genetic makeup, are the key factors that influence this complex disease. Recently, we identified haplotypes in the IL4 gene that were associated with chronic periodontitis (CP). This study aimed to evaluate whether subjects with different IL4 haplotypes (TCI/CCI and TTD/CTI) would be differentially colonized by periodontopathogens and whether they would respond differently to non-surgical periodontal therapy. Thirty-nine patients carrying the IL4 haplotype of genetic susceptibility to CP (IL4+) or protection against CP (IL4-) were evaluated. Those groups were further subdivided into individuals with CP (CP IL4+ or CP IL4-) and those that were periodontally healthy (H) (H IL4+ or H IL4-). CP patients were submitted to non-surgical periodontal therapy. Clinical and microbiological analyses were performed considering the data at baseline and 45 and 90 days after periodontal therapy. Periodontopathogens levels were evaluated by absolute quantitative polymerase chain reaction (qPCR). The baseline data revealed that the total levels of periodontopathogens were higher in the CP IL4+ than in the CP IL4- groups. Clinical analyses revealed that the periodontal therapy was equally effective, independent of the subject's IL4 genetic load. The TCI/CCI IL4 haplotype, previously associated with genetic susceptibility to CP, was also associated with increased levels of periodontopathogenic bacteria, but this genetic background did not influence the response to non-surgical periodontal treatment.

Stage-specific gene expression during spermatogenesis in the Senegalese sole (Solea senegalensis), a fish with semi-cystic type of spermatogenesis, as assessed by laser capture microdissection and absolute quantitative PCR

Spermatogenesis is a complex process where hormonal signals regulate the interaction of different cell types in a tight spatial and temporal fashion. The Senegalese sole (Solea senegalensis) is a marine flatfish that, in contrast to many fish, exhibits a semi-cystic, asynchronous pattern of spermatogenesis progression. This pattern is characterized by the release of spermatids into the tubule lumen, where they transform into spermatozoa. In this study, we used laser capture microdissection (LCM) to isolate cells from cysts containing spermatogonia, spermatocytes, spermatids or spermatozoa in order to investigate developmental patterns of gene expression. Furthermore, we also analyzed the stage-specific expression of the same set of genes throughout spermatogenesis (early-mid, late and maturing spermatogenic stages) in tissue fragments of the Senegalese sole testis. Genes analyzed by absolute qPCR in cysts isolated by LCM and stage-specific testis samples included genes involved in steroid synthesis and action (3β-hsd, 17β-hsd, 20β-hsd, star, star-like, progesterone receptor), gonadotropin action (fshr, lhr), the kisspeptin system (kiss2, kiss2r) and other genes important for the production of mature gametes (zona pellucida 2.2, claudin and clusterin). Our results show that, in general, steroidogenesis-related genes tended to increase with spermatogenesis progression and that 3β-hsd and 20β-hsd were expressed in germ cells but 17β-hsd was not. Our results also show that fshr is expressed in most testicular cell types, including germ cells. In contrast, lhr is expressed only in late spermatogenesis and is not expressed in any of the germ cell types examined, indicating that, in contrast to fshr, lhr may be primarily expressed in non-germinal cells (e.g. Leydig cells). Furthermore, kisspeptin and its receptor were expressed in all germ cell types examined and, as expected, gamete maturation-related genes were more expressed in mature stages. These results illustrate that key factors that participate in the hormonal regulation of spermatogenesis in the Senegalese sole testis show complex cell type- and stage-specific patterns of gene expression.

Transport Function and Transcriptional Regulation of a Liver-Enriched Human Organic Anion Transporting Polypeptide 2B1 Transcriptional Start Site Variant

Human organic anion transporting polypeptide 2B1 (OATP2B1) is a membrane transporter that facilitates the cellular uptake of a number of endogenous compounds and drugs. OATP2B1 is widely expressed in tissues including the small intestine, liver, kidney, placenta, heart, skeletal muscle, and platelets. It was recently shown that differential promoter usage in tissues results in expression of five OATP2B1 transcriptional start site variants that use distinct first exons but share common subsequent exons. These variants are expected to encode either a full-length (OATP2B1-FL) or shortened protein lacking 22 N terminus amino acids (OATP2B1-Short). Little is known regarding the transport activity and regulation of OATP2B1 variants with N terminus truncation. Here, using absolute quantitative polymerase chain reaction, we find the full-length variant is the major form expressed in duodenum but the short variant predominates in liver. Using a transient heterologous cell expression system, we find that the transport activities of the short OATP2B1 variant toward substrates estrone sulfate and rosuvastatin are similar to the well characterized full-length variant. Transcriptional activity screening of the liver-enriched OATP2B1 variant promoter identified hepatocyte nuclear factor 4 α (HNF4α) as a novel transacting factor. With a combination of in silico screening, promoter mutation in cell-based reporter assays, small interfering RNA knockdown, and chromatin immunoprecipitation (ChIP) studies, we identified a functional HNF4α binding site close to the transcription start site (-17 to -4 bp). We conclude that the major OATP2B1 protein form in liver is transport competent and its hepatic expression is regulated by HNF4α.

The human L-type calcium channel Cav1.3 regulates insulin release and polymorphisms in CACNA1D associate with type 2 diabetes

Voltage-gated calcium channels of the L-type have been shown to be essential for rodent pancreatic beta cell function, but data about their presence and regulation in humans are incomplete. We therefore sought to elucidate which L-type channel isoform is functionally important and its association with inherited diabetes-related phenotypes. Beta cells of human islets from cadaver donors were enriched using FACS to study the expression of the genes encoding voltage-gated calcium channel (Cav)1.2 and Cav1.3 by absolute quantitative PCR in whole human and rat islets, as well as in clonal cells. Single-cell exocytosis was monitored as increases in cell capacitance after treatment with small interfering (si)RNA against CACNA1D (which encodes Cav1.3). Three single nucleotide polymorphisms (SNPs) were genotyped in 8,987 non-diabetic and 2,830 type 2 diabetic individuals from Finland and Sweden and analysed for associations with type 2 diabetes and insulin phenotypes. In FACS-enriched human beta cells, CACNA1D mRNA expression exceeded that of CACNA1C (which encodes Cav1.2) by approximately 60-fold and was decreased in islets from type 2 diabetes patients. The latter coincided with diminished secretion of insulin in vitro. CACNA1D siRNA reduced glucose-stimulated insulin release in INS-1 832/13 cells and exocytosis in human beta cells. Phenotype/genotype associations of three SNPs in the CACNA1D gene revealed an association between the C allele of the SNP rs312480 and reduced mRNA expression, as well as decreased insulin secretion in vivo, whereas both rs312486/G and rs9841978/G were associated with type 2 diabetes. We conclude that the L-type calcium channel Cav1.3 is important in human glucose-induced insulin secretion, and common variants in CACNA1D might contribute to type 2 diabetes.

Integration Frequency and Intermolecular Recombination of rAAV Vectors in Non-human Primate Skeletal Muscle and Liver

The comprehensive characterization of recombinant adeno-associated viral (rAAV) integration frequency and persistence for assessing rAAV vector biosafety in gene therapy is severely limited due to the predominance of episomal rAAV vector genomes maintained in vivo. Introducing rAAV insertional standards (rAIS), we show that linear amplification-mediated (LAM)-PCR and deep sequencing can be used for validated measurement of rAAV integration frequencies. Integration of rAAV2/1 or rAAV2/8, following intramuscular (IM) or regional intravenous (RI) administration of therapeutically relevant vector doses in nine adult non-human primates (NHP), occurs at low frequency between 10(-4) and 10(-5) both in NHP liver and muscle, but with no preference for specific genomic loci. High resolution mapping of inverted terminal repeat (ITR) breakpoints in concatemeric and integrated vector genomes reveals distinct vector recombination hotspots, including large deletions of up to 3 kb. Moreover, retrieval of integrated rAAV genomes indicated approximately threefold increase in liver compared to muscle. This molecular analysis of rAAV persistence in NHP provides a promising basis for a reliable genotoxic risk assessment of rAAV in clinical trials.

FOXM1 Induces a Global Methylation Signature That Mimics the Cancer Epigenome in Head and Neck Squamous Cell Carcinoma

The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16INK4A promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant FOXM1 expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with in vivo mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique FOXM1-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions.

Expression of HERV-Fc1, a Human Endogenous Retrovirus, Is Increased in Patients with Active Multiple Sclerosis

Multiple sclerosis (MS) is considered to be an autoimmune disease with an unknown cause and with immune system dysregulation. Among environmental factors, viruses are most often connected with the etiology of MS. Human endogenous retroviruses (HERVs) constitute 5 to 8% of human genomic DNA and have been detected as transcripts and proteins in the central nervous system (CNS) and peripheral blood, frequently in the context of neuroinflammation. HERV-Fc1, which belongs to the HERV-H/F family, has received our attention largely because of the genetic association with MS. We studied the expression of a capsid (Gag) protein of HERV-H/F origin by flow cytometry in peripheral blood mononuclear cells (PBMCs) from healthy controls and from MS patients with nonactive or active disease. There was a significant increase in HERV-H/F Gag expression in CD4(+) (P < 0.001) and CD8(+) (P < 0.001) T lymphocytes and in monocytes (P = 0.0356) in PBMCs from MS patients with active disease. Furthermore, we have undertaken the first rigorous SYBR green-based absolute quantitative PCR (Q-PCR) evaluation approach to quantify extracellular HERV-Fc1 RNA viral loads in plasma from MS patients and healthy controls. We found a 4-fold increase in extracellular HERV-Fc1 RNA titers in patients with active MS compared with healthy controls (P < 0.001). These findings strengthen the link between HERV-Fc1 and the pathology of MS. The cause and biological consequences of these differential expression levels will be the subject of further investigation. HERV-Fc1 biology could be a compelling area for understanding the pathology of MS and possibly other autoimmune disorders.

Endothelial colony-forming cells show a mature transcriptional response to shear stress

Endothelial progenitor cells (EPC) play a central role in endothelial maintenance and repair. Endothelial colony-forming cells (ECFC) form a subpopulation of EPC. ECFC are readily attainable, can be easily isolated, possess a high proliferation potential, and are therefore a promising source of endothelial cells (EC) for future cardiovascular therapeutic applications. The extent to which these cells respond to shear stress as adult vascular EC remains to be elucidated. Here, we study the transcriptional response of ECFC induced by shear stress and compare it with the response of mature arterial and venous cells. ECFC, as well as human umbilical vein EC (HUVEC) and human umbilical artery EC (HUAEC), were subjected to low (0.5Pa) and high (2.5Pa) shear stress. The endothelial differentiation phenotype and transcriptional responses were analyzed using immunocytochemistry and quantitative polymerase chain reaction (Q-PCR). Performing absolute quantification of copy numbers by Q-PCR allows comparing the responses of cell types relative to each other. Our data show that isolated ECFC resemble mature EC in cobblestone morphology and endothelial marker expression. Absolute Q-PCR quantification revealed that although being truly endothelial, ECFC do not fully resemble HUVEC or HUAEC in the expression of specific differentiation markers. When subjected to shear stress, ECFC show a mature response to fluid flow, comparable to that of HUVEC and HUAEC. The capacity of endothelial progenitors to respond to fluid flow in a similar manner to HUVEC and HUAEC highlights the universal response of EC to fluid shear stress, independently of their endothelial differentiation status. This property supports the use of these cells as an EC source for tissue engineering applications.

Natural co-infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) and infectious myonecrosis virus (IMNV) in Litopenaeus vannamei in Brazil

Cultivation of whiteleg shrimp ( Litopenaeus vannamei) constitutes a growing aquaculture industry in northeast Brazil. Similar to other animals that are intensively farmed, shrimp experience disease outbreaks, which are a constant threat and cause eventually significant economical losses. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) and infectious myonecrosis virus (IMNV) are prevalent epizootic viral agents in Brazil. In a routine monitoring program for the diagnosis of IHHNV and IMNV, using molecular techniques like conventional PCR, reverse transcription coupled with PCR (RT-PCR) and absolute quantitative PCR (qPCR), we found that most positive samples of shrimp were simultaneously co-infected with both viruses. This survey is the first to show the occurrence of a natural co-infection that is caused by IHHNV and IMNV in penaeid shrimp attacked by viral disease that were cultivated in Northeast Brazil.

First Line Treatment with Nilotinib 800 Mg Daily Results In Unprecedentedly High Rate of Rapid, “Deep” and Stable Molecular Responses as Assessed by a High Sensitive Nanofluidic Array for the Detection of Rare Copies of BCR-ABL1 Transcript: Results of a Phase 2 Trial of the GIMEMA CML Working Party

AbstractAbstract 2720Treatment strategies based on second generation tyrosine kinase inhibitors such as Nilotinib, have improved overall chronic myeloid leukemia (CML) treatment results, with a rapid and major molecular remission (MCR), at 12 months rate ranging 75%. Therefore, the detection of residual leukemic cells by measuring BCR-ABL1 transcript level is absolutely required to monitor minimal residual disease. To investigate the molecular therapeutic efficacy of nilotinib 400 mg BID in previously untreated, ECP, Philadelphia-positive CML patients, the Italian GIMEMA CML Working Party is conducting an multicenter phase II study (ClinicalTrials.gov NCT00481052). The primary endpoint is the CCgR rate at 1 year. The kinetic of molecular response is being studied by Q-PCR at baseline and after 1, 2, 3, 6, 9, 12, 18 and 24 months from treatment start by a TaqMan absolute quantitative PCR (Applied Biosystems 7900HT Fast Real-Time PCR System) and by the 12.765 Digital array (Fluidigm) which is is a nanofluidic biochip consisting in twelve panels, each containing 765 individual reaction chambers of 6 nL volume. Fluidigm analysis was done in parallel on same samples (at diagnosis,1, 2, 3, 6, 9, 12, and were possible at 18 and 24 months). Patient with longest follow up was 727 days. A Major Molecular Response, defined as a BCR-ABL:ABL ratio <0.1% according to the International Scale, and obtained by conventional RT-PCR was already achieved in 52% at 3 months, 66% at 6 months 73% at 9 months and 85% at 12 months. The median BCR-ABL transcripts level at 3, 6, 9 and 12 months was 0.063, 0.018, 0.018 and 0.006, respectively. On 73 patients with the longest follow up, a complete molecular response (CMR) defined as a BCR-ABL:ABL ratio <0.0032%IS and a negative nested PCR, was achieved in 7 patients at 12 months. The log-reduction in BCR-ABL/ABL level at 3 months was predictive of the probability of MMR at 12 months. Probability to obtain a MMR at 12 months was of 42%, 62% and 95%, respectively.The molecular results achieved in our study strongly support the notion that in early chronic phase CML patients molecular responses to nilotinib are substantially faster and “deeper” than those to IM. More rapid reduction of residual disease might help to reduce failures and to improve the late outcome of therapy.Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures:No relevant conflicts of interest to declare.

Use of a High Sensitive Nanofluidic Array for the Detection of Rare Copies of BCR-ABL1 Transcript In Patients with Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL).

AbstractAbstract 1677The BCR-ABL1 positive ALL is the most frequent and prognostically most unfavorable subtype of ALL in adults. Treatment strategies based on tyrosine kinase inhibitors of first and second generation have improved overall treatment results, with a rapid and complete remission (CR) rate ranging 90%. Nevertheless most patients experienced hematological relapse in a short time, also after hematopoietic stem cell transplantation. Therefore, the detection of residual leukemic cells by measuring BCR-ABL1 transcript level is absolutely required to monitor minimal residual disease and to detect early relapse. Different assays based on quantitative polymerase chain reaction (qPCR) are available and they vary in respect to the type of instrument used, the primer and probe location, the real-time chemistry and the control gene used. Differences among methods can lead to a variation in the sensitivity and measurement reliability. In order, to investigate the efficacy of a high sensitive method based on nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify residual and rare BCR-ABL1 copies, we compared the molecular results obtained by conventional qPCR with those obtained by nanofluidic Fluidigm approach. At the time of writing, 60 BCR-ABL1-positive ALL samples (42 positive for the p190 BCR-ABL1 isoform and 18 for the p210) were analyzed. First, we performed a TaqMan absolute quantitative PCR (Applied Biosystems 7900HT Fast Real-Time PCR System); RNA integrity was evaluated using the control gene ABL1. Both BCR-ABL1 or ABL1 copy number copies were derived by the interpolation of cycle threshold (Ct) values to the appropriate standard curve obtained using different plasmid dilutions (Ipsogen), each containing known BCR-ABL1 or ABL1 gene copies. Samples resulted in complete (38.3%) or major (61.7%) molecular response (BCR-ABL1/ABL1 ratio ≤ 0.001 or < 0.1, respectively and according to the International Scale). Subsequently, we quantified again the samples using the 12.765 Digital array (Fluidigm). This is a nanofluidic biochip that consists in twelve panels, each containing 765 individual reaction chambers of 6 nL volume. Samples are portioned prior to qPCR into the single chambers of the panel; as fluorescent signal is produced only in chambers containing copies of the target sequence, digital array provides an absolute quantification by counting the number of positive reactions. Following amplification, digital raw data are processed by the BioMark Digital PCR Analysis software (Fluidigm), that estimates the true number of molecules per chamber using the Poisson probabilistic distribution. First, we assessed the sensitivity and reproducibility of the assay using six serial dilutions of plasmids (Ipsogen) expressing known copy number of BCR-ABL1 p190 transcript (10000; 1000; 100; 50; 10; 1 copies). A 2 μl volume of input leukemia cDNA was loaded and two panels for each dilution were used. Analysis parameters chosen for digital raw data processing were automated set threshold and target Ct range 20–40. Results showed a detection rate until a copy of target sequence (Fig. 1) and a pairing significantly effective between replicates. We then analyzed duplicates of BCR-ABL1 positive ALL samples with a positive control for each chip: digital array resulted positive in 22 major molecular response samples (59.46%; 3.25 median number of copies detected, range 0.5–33.5), and in 1 complete molecular response sample (4.34%). Moreover, the comparison between TaqMan and Fluidigm methods resulted in the detection of the same BCR-ABL1 copies in 53.34% (group I); in 35.0% the quantity detected by Fluidigm was lower than that detected by TaqMan (group II); finally, in 11.66% the Fluidigm approach resulted more sensitive than TaqMan (group III). In conclusion, we demonstrated that the Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript, as demonstrated with plasmid serial dilution detection rate and with samples in molecular remission and it could provide an accurate monitoring method for BCR-ABL1-positive ALL CR patients. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. [Display omitted] Disclosures:Baccarani:NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

Epstein–Barr virus DNA load in tumour tissues correlates with poor differentiation status in non‐muscle invasive urothelial carcinomas

To detect the correlation between the clinical staging, grading and genomic Epstein-Barr virus (EBV) viral numbers in tumour tissues of urothelial carcinoma patients. From June 2004 to May 2008, 60 urothelial carcinoma patients (50 cases of bladder carcinoma and 10 of upper tract urothelial carcinoma (UTUC) were enrolled in the study. Eight patients who underwent transurethral resection of prostate for prostate hyperplasia and two patients receiving nephrectomy for non-function kidney were used as normal controls. The EBV viral copy numbers in genomic DNA were evaluated using a real-time PCR-based study. The BamHI W region of the Namalwa cell line was constructed to the plasmid clone and was used as standard curve for absolute quantitative PCR (Q-PCR). Epstein-Barr virus DNA was detected in 56% (28/50) and 60% (6/10) of the bladder and UTUC patients, respectively. The EBV DNA could not be detected in the normal control group. By pooling the UTUC and bladder patients in stage Ta,T1, the high copy number in fixed genomic DNA amount (100 ng/20 µL) was correlated with the high grading in stage Ta,T1 urothelial carcinoma (P = 0.014). The overall grading was not statistically associated with EBV copy number (P = 0.25). Although the copy numbers between paired tumour and normal tissues were not statistically different (P= 0.169), there were more copies of EBV in the normal tissues adjacent to the tumours than in those free from urothelial carcinoma. There was no significant difference between recurrence of non-muscle invasive bladder cancer and the presence of EBV (P > 0.05). Epstein-Barr virus DNA could be detected in the genome of the urothelial carcinoma specimens. The poor differentiation status was correlated with the high load of the EBV genome in non-muscle invasive urothelial carcinoma. However, recurrence-free survival was not greater in EBV-positive patients than in EBV-negative patients.

Hormonally regulated overexpression of Arabidopsis WUS and conifer LEC1 (CHAP3A) in transgenic white spruce: implications for somatic embryo development and somatic seedling growth

Adult conifers are still recalcitrant in clonal propagation despite significant advances in forest tree biotechnology. Plant regeneration through somatic embryogenesis from explants older than mature zygotic embryos is either difficult or impossible to achieve. To investigate if ectopic expression of transcription factors involved in the induction of the embryogenic process would induce somatic embryogenesis in Picea glauca (white spruce) somatic plants, we used the LEAFY-COTYLEDON1 homolog cloned from Picea mariana, CHAP3A, and Arabidopsis thaliana WUS to transform embryonal mass of P. glauca. Ectopic gene expression was induced by 17-beta-estradiol during stages of somatic embryogenesis (early embryogenesis and late embryogenesis) and somatic seedling growth in the transgenics. Of the two transcription factors, only WUS produced severe phenotypes by disrupting the development of somatic embryos on the maturation medium and inhibiting germination. However, none of the transgenes induced ectopic somatic embryogenesis even in the presence of plant growth regulators. Absolute quantitative PCR confirmed the expression of both CHAP3A and WUS in transgenic embryonal mass and in all parts of somatic seedlings. A high expression of the transgenes did not influence expression profiles of any of the ten other transcription factors tested, some of which have been known to be involved in the process of embryogenesis. Implications of these results for further work are discussed.