Multiplex RT-qPCR Research Articles

Evaluation of the prognostic significance of RACGAP1, Ki67, and TOP2A mRNA expression in high-risk early breast cancer.

582 Background: Proliferation is a major process in carcinogenesis. RACGAP1 is a protein involved in cell growth regulation and metastasis, Ki67 is a known proliferation marker and TOP2A has a key role in DNA replication and remodeling. The aim of the present study was to explore the prognostic significance of a signature of proliferation markers, such as RACGAP1, Ki67 and TOP2A on disease-free survival (DFS) and overall survival (OS) in high-risk early breast cancer patients. Methods: A total of 1681high-risk breast cancer patients, enrolled in two consecutive phase III trials, were treated with anthracycline-based adjuvant chemotherapy. Formalin-fixed paraffin-embedded tumor tissue samples from 963 of these patients were extracted using a standardized fully automated isolation method for total RNA based on silica-coated magnetic beads, followed by multiplex RT-qPCR for assessing RACGAP1, Ki67 and TOP2A mRNA expression. CALM2 was included in the same reaction, as a reference gene. Results: After a median follow-up of 107 months, 289 patients (30.0%) demonstrated disease progression and 261 (27.1%) patients died. Univariate analysis revealed that poor OS was associated with high RACGAP1 mRNA expression (p=0.0185, log-rank), high Ki67 (p=0.0219), as well as high TOP2A (p=0.0019) mRNA expression, while in multivariate analysis only TOP2A retained significance (Wald’s p=0.008). In an effort to improve prognostic significance, combinations of the expression of two or all three genes were tested, with low mRNA expression of the three genes being associated with improved DFS (HR=0.74, CI=0.56-0.98, p=0.035) and OS (HR=0.60, CI=0.42-0.85, p=0.004). However, in multivariate analysis, none of the combinations retained prognostic significance, except the combination of high RACGAP1 and TOP2A mRNA expression, which was found to be associated with decreased DFS (HR=1.26, CI=0.96-1.63, p=0.092) and OS (HR=1.49, CI=1.10-2.03, p=0.009). Conclusions: High RACGAP1 and TOP2A mRNA expression was found, in multivariate analysis, to be of adverse prognostic significance in high-risk early breast cancer patients treated with anthracycline-containing adjuvant chemotherapy.

Association of mesenchymal phenotype in circulating tumor cells with poor prognosis in metastatic colorectal cancer.

e14567 Background: Although the prognostic relevance of circulating tumor cells (CTCs) is well established in metastatic colorectal cancer (mCRC), their clinical use is currently constrained by the phenotypic definition of such cells that is largely limited to epithelial markers. Our objective in this study is to characterize the prognostic significance of a larger group of CTCs including those with characteristics of epithelial-mesenchymal transition (EMT) or cancer stem cells (CSCs) in a cohort of pre-treated mCRC patients (pts). Methods: Peripheral blood was collected in AdnaCollect preservation tubes from pts for immunomagnetic tumor cell enrichment using the AdnaTest ColonCancer Select kit and multiplex qRT-PCR analyses of tumor-associated epithelial (CEA, EPCAM, EGFR), cancer stem cell (ALDH1) and mesenchymal (TWIST1, AKT2, PIK3CA) transcripts. Results: In the initial phase, 41 previously treated mCRC pts (median prior therapies 3) were enrolled over a 4 month period and were followed prospectively. In this cohort, there was a low rate of CEA, EPCAM, and EGFR expression in the CTCs (12%, 2%, and 0%, respectively), consistent with loss of epithelial markers. Conversely, ALDH1, an established CSC marker was expressed in 30% of pts, with only one pt’s CTCs expressing both ALDH1 and CEA expression. ALDH1 expression was associated with very poor prognosis (HR 24.6, p=0.037) and 57% 60-day mortality, compared to 0% 60-day mortality for pts with no detectable ALDH1 expression. The mesenchymal markers of TWIST1, AKT2, and PIK3CA were strongly correlated with ALDH1 expression (Spearman r=0.55, 0.59, 0.63, respectively, p < 0.01 for each). Conclusions: The majority of CTCs detected in refractory mCRC pts have lost epithelial markers. Acquisition of markers associated with EMT or CSCs appear to be associated with poor prognosis, and may provide important insights into better prognostication and also biology of refractory colorectal cancer. Further enrolment into this study is ongoing and updated results will be presented.

Characterization of the function and co-expression patterns of human FOXP3 isoforms and their role in IPEX syndrome development. (P4146)

Abstract IPEX syndrome is a rare and fatal X-linked recessive disorder due to mutations in the FOXP3 gene, which plays a critical role in the development and function of the regulatory T cells (Tregs) that maintain self-tolerance. Contrary to mice, humans possess three isoforms of FOXP3 with unknown function, and the expression of these isoforms is not restricted to Tregs. We determined the levels of expression and distribution of FOXP3 and other transcription factors isoforms in normal human T-cell subsets and in T cell subsets of patients with autoimmunity at the single-cell level by multiplex RT-qPCR and Flow cytometry and observed that expression of these isoforms is not restricted to Tregs, but can also be detected in naïve and activated/memory helper (Th) and in activated/memory CD8+ T cells. Distinct patterns of co-expression of the FOXP3 isoforms are modulated in response to inflammatory or autoimmune stimuli. Moreover, we observed that normal FOXP3 isoforms could induce bona fide regulatory function in Th cells. Our work supports the view that lack of FOXP3 expression in the effector T-cell populations in IPEX patients may contribute to the pathogenesis of the disease and suggests that FOXP3 deficiency can be corrected by gene transfer of the appropriate FOXP3 isoforms into conventional T-cells, which has important implication for the treatment of IPEX patients.

Abstract 446: Differential expression of immuno-regulatory genes associated with PD-L1 display: Implications for clinical blockade of the PD-1/PD-L1 pathway in melanoma.

Abstract Background: Blockade of the immunosuppressive PD-1/PD-L1 pathway has shown promising clinical results in patients with treatment-refractory solid tumors including melanoma. PD-L1, a protein displayed by many tumors, ligates the PD-1 co-inhibitory receptor on activated T cells. We previously found that IFN-γ, a potent inducer of PD-L1, was expressed by TILs in PD-L1(+) but not PD-L1(-) melanomas, creating an immunosuppressive microenvironment by a mechanism that we term “adaptive immune resistance” (Taube et al., Science Transl Med 2012). In the current study, we assessed other factors associated with PD-L1 expression in melanoma through differential gene expression analysis, in order to better understand the biology of this pathway and identify potential targets for combination immunotherapies. Methods: PD-L1(+) vs. (-) melanomas including immune cell infiltrates (n=11) were laser-capture microdissected (LCM) from formalin-fixed paraffin embedded (FFPE) specimens and analyzed by whole genome array analysis with cDNA-mediated Annealing, Selection, extension and Ligation (DASL), a novel technology that allows for gene expression analysis with partially degraded mRNAs obtained from FFPE specimens. Differentially expressed genes were submitted to the NIH functional analysis tool DAVID to search for enrichment in biologically related groups. Multiplex quantitative (q)RT-PCR was used to validate differential expression of genes of interest and to examine other candidate genes in new set of PD-L1(+) vs.(-) melanomas (n=11). Results: DASL/DAVID analysis of PD-L1(+) vs. PD-L1(-) melanomas uncovered genes of interest in several immunologically relevant pathways, including “Defense Response” (p<1.5E-9) and “T-cell activation” (p<2.2E-5). Multiplex qRT-PCR to examine genes of interest from the DAVID analysis along with other potentially relevant candidate genes was normalized to the GUSB housekeeping gene or to CD45 (pan-leukocyte marker), revealing over-expression of genes associated with immunosuppression (PD-1, PD-L1, LAG-3, IL-10), activated CD8 T cells (CD8A, IFN-γ, lysozyme, perforin, CCL5/RANTES), and antigen presenting cells (CD163, TLR3, CXCL1) in PD-L1(+) melanomas (p<0.10). Conclusions: These experiments identified groups of functionally related, differentially expressed immuno-regulatory genes in PD-L1(+) melanomas. These factors may coordinately create an immunosuppressive tumor microenvironment, by synergizing to enhance PD-L1 expression on tumor cells and to inhibit T cell function. Immunohistochemical and in vitro functional validation assays of candidate gene products are currently underway in order to identify new ways to overcome adaptive immune resistance in synergistic treatment combinations with PD-1/PD-L1 blockade. Citation Format: Geoffrey D. Young, Tracee L. McMiller, Haiying Xu, Shuming Chen, Alan E. Berger, Jinshui Fan, Robert A. Anders, Christopher Cheadle, Drew M. Pardoll, Suzanne L. Topalian, Janis M. Taube. Differential expression of immuno-regulatory genes associated with PD-L1 display: Implications for clinical blockade of the PD-1/PD-L1 pathway in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 446. doi:10.1158/1538-7445.AM2013-446

Comparison of three molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer

IntroductionComparison studies between different analytical methodologies for circulating tumor cells (CTC) detection and molecular characterization are urgently needed, since standardization of assays is essential before their use in clinical practice.MethodsWe compared three different CTC molecular assays. To avoid discrepancies due to pre-analytical errors we used the same cDNAs throughout our study. CTC were isolated using anti-EpCAM and anti-MUC1 coated magnetic beads from 2 × 5 ml of peripheral blood of 254 early and 51 metastatic breast cancer patients and 30 healthy individuals. The same cDNAs were analyzed by: a) singleplex RT-qPCR assay for CK-19; b) multiplex RT-qPCR for CK-19, HER-2, MAGE- A3, and PBGD; and c) a commercially available molecular assay (AdnaTest BreastCancer) for GA733-2, MUC-1, HER-2 and beta-actin.ResultsIn early breast cancer, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest, were positive for the presence of CTC in 14.2%, 22.8% and 16.5% subjects, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 72.4% while between the AdnaTest and multiplex RT-qPCR was 64.6%. In patients with overt metastasis, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest were positive in 41.2%, 39.2% and 54.9% patients, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 70.6% while between the AdnaTest and multiplex RT-qPCR was 68.6%.ConclusionsAll CTC assays gave similar results in about 70% of cases. Better agreement was found in the metastatic setting, possibly explained by the higher tumor load in this group. Discordances could be attributed to the different gene transcripts used to evaluate CTC positivity. Our results indicate the importance of CTC heterogeneity for their detection by different analytical methodologies.

A9.17 Targeting the Expression of miR-146a in Mouse Inflammatory Ly6Chigh Monocyte Subset for Therapeutic Intervention in Arthritis

Background and ObjectivesMonocytes can give rise to different cell types including osteoclasts (OC), which play an important role in maintaining bone homeostasis by resorbing bone matrix. Circulating monocytes consist of...

Single-Tube Multiplexed Molecular Detection of Endemic Porcine Viruses in Combination with Background Screening for Transboundary Diseases

Detection of several pathogens with multiplexed real-time quantitative PCR (qPCR) assays in a one-step setup allows the simultaneous detection of two endemic porcine and four different selected transboundary viruses. Reverse transcription (RT)-qPCR systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), two of the most economically important pathogens of swine worldwide, were combined with a screening system for diseases notifiable to the World Organization of Animal Health, namely, classical and African swine fever, foot-and-mouth disease, and Aujeszky's disease. Background screening was implemented using the identical fluorophore for all four different RT-qPCR assays. The novel multiplex RT-qPCR system was validated with a large panel of different body fluids and tissues from pigs and other animal species. Both reference samples and clinical specimens were used for a complete evaluation. It could be demonstrated that a highly sensitive and specific parallel detection of the different viruses was possible. The assays for the notifiable diseases were even not affected by the simultaneous amplification of very high loads of PRRSV- and PCV2-specific sequences. The novel broad-spectrum multiplex assay allows in a unique form the routine investigation for endemic porcine pathogens with exclusion diagnostics of the most important transboundary diseases in samples from pigs with unspecific clinical signs, such as fever or hemorrhages. The new system could significantly improve early detection of the most important notifiable diseases of swine and could lead to a new approach in syndromic surveillance.

Forensic Body Fluid Identification by Analysis of Multiple RNA Markers Using NanoString Technology

RNA analysis has become a reliable method of body fluid identification for forensic use. Previously, we developed a combination of four multiplex quantitative PCR (qRT-PCR) probes to discriminate four different body fluids (blood, semen, saliva, and vaginal secretion). While those makers successfully identified most body fluid samples, there were some cases of false positive and negative identification. To improve the accuracy of the identification further, we tried to use multiple markers per body fluid and adopted the NanoString nCounter system instead of a multiplex qRT-PCR system. After measuring tens of RNA markers, we evaluated the accuracy of each marker for body fluid identification. For body fluids, such as blood and semen, each body fluid-specific marker was accurate enough for perfect identification. However, for saliva and vaginal secretion, no single marker was perfect. Thus, we designed a logistic regression model with multiple markers for saliva and vaginal secretion and achieved almost perfect identification. In conclusion, the NanoString nCounter is an efficient platform for measuring multiple RNA markers per body fluid and will be useful for forensic RNA analysis.

In vitro evaluation of cytochrome P450 induction and the inhibition potential of mitragynine, a stimulant alkaloid

CYP450 enzymes are key determinants in drug toxicities, reduced pharmacological effect and adverse drug reactions. Mitragynine, an euphoric compound was evaluated for its effects on the expression of mRNAs encoding CYP1A2, CYP2D6 and CYP3A4 and protein expression and resultant enzymatic activity. The mRNA and protein expression of CYP450 isoforms were carried out using an optimized multiplex qRT-PCR assay and Western blot analysis. CYP1A2 and CYP3A4 enzyme activities were evaluated using P450-Glo™ assays. The effects of mitragynine on human CYP3A4 protein expression were determined using an optimized hCYP3A4–HepG2 cell-based assay. An in silico computational method to predict the binding conformation of mitragynine to the active site of the CYP3A4 enzyme was performed and further validated using in vitro CYP3A4 inhibition assays. Mitragynine was found to induce mRNA and protein expression of CYP1A2. For the highest concentration of 25μM, induction of mRNA was approximately 70% that of the positive control and was consistent with the increased CYP1A2 enzymatic activity. Thus, mitragynine is a significant in vitro CYP1A2 inducer. However, it appeared to be a weak CYP3A4 inducer at the transcriptional level and a weak CYP3A4 enzyme inhibitor. It is therefore, unlikely to have any significant clinical effects on CYP3A4 activity.

Identification of Novel Adult Mesenchymal Progenitors That Can Contribute to HSC Niche

AbstractAbstract 3467The microenvironment (niche) cues hematopoietic stem cell (HSC) receives play an important role in the regulation of their decisions between self-renewal and differentiation. However, the cellular constitution of niches remains poorly understood. We identified three adult progenitor populations near the endosteum based on differential expression of cell surface markers, including CD166, CD146 and Sca1. Upon co-transplantation with fetal skeletal progenitors, Sca1+ progenitors can give rise to CD146+ and CD166+ stroma and phenotypically CXCL12 abundant reticular (CAR) cells in marrow. CD146+ and CD166+ progenitors, on the other hand, form bone without marrow cavity. Multiplex single cell qRT-PCR reveals all three progenitors expressed high levels of genes involved in HSC maintenance. In vitro co-culture assay demonstrated that all three progenitors could preserve HSC long-term multi-lineages reconstitution capability. Furthermore, disruption of stem cell factor (SCF) production in Sca1+ progenitor severely limits its ability to support HSC both in culture condition and after transplantation. Our results suggested that Sca1+, CD146+, and CD166+ mesenchymal progenitors and their progeny collaborate to provide supportive environment for hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Changes of proteome and phosphoproteome trigger embryo–larva transition of honeybee worker (Apis mellifera ligustica)

The development of the last day embryo to the first instar larva is an essential process in the honeybee life cycle. However, the molecular mechanism of this life transition is still unknown. The proteome and phosphoproteome of last day embryos (72 h) and first instar larvae (24h, post hatching) were analyzed using 2-DE, multiplex fluorescent staining, mass spectrometry, bioinformatics, and qRT-PCR. Sixty-five proteins and 34 phosphoproteins changed their expression across the shift of embryos to larvae. The embryo stronger expression of proteins related to energy metabolism, development and amino acid metabolism suggests its high metabolic energy demand during active embryogenesis. While, the newly hatched larvae escalated the expression of proteins related to cytoskeleton, biosynthesis, protein folding, fatty acid and oxidative metabolism, particularly the higher phosphorylation of cytoskeleton and biosynthesis indicates their roles to ensure the fast growing larvae. These differences in protein expression level illustrate that specific protein functions are restricted to particular developmental stage. Our data suggest the essential changes of proteome and phosphoproteome to trigger the transition of embryo to larvae. This unravels the molecular event behind the first life cycle transition of honeybees and is potentially helpful for future reverse genetic studies in this model insect.

Genome-wide mRNA profiling and multiplex quantitative RT-PCR for forensic body fluid identification

In forensic science, identifying a tissue where a forensic specimen was originated is one of the principal challenges. Messenger RNA (mRNA) profile clearly reveals tissue-specific gene expression patterns that many attempts have been made to use RNA for forensic tissue identification. To systematically investigate the body-fluid-specific expression of mRNAs and find novel mRNA markers for forensic body fluid identification, we performed DNA microarray experiment with 24 Korean body fluid samples. Shannon entropy and Q-values were calculated for each gene, and 137 body-fluid-specific candidate genes were selected. By applying more stringent criteria, we further selected 28 candidate genes and validated them by RT-PCR and qRT-PCR. As a result, we suggest a novel combination of four body-fluid-specific mRNA makers: PPBP for blood, FDCSP for saliva, MSMB for semen and MSLN for vaginal secretion. Multiplex qRT-PCR assay was designed using the four mRNA markers and DNA/RNA co-extraction method was tested for forensic use. This study will provide a thorough examination of body-fluid-specifically expressed mRNAs, which will enlarge the possibility of practical use of RNA for forensic purpose.

Comparison of immunohistochemistry (IHC) and quantitative RT-PCR: ER, PR, and HER2 receptor status.

47 Background: IHC is the present standard for measuring estrogen (ER) and progesterone receptor (PR) expression for breast cancer. However, the lack of concordance between testing laboratories and the critical patient treatment decisions made with results prompted ASCO/CAP to recommend guidelines and proficiency testing requirements to ensure accuracy. Quantitative RT-PCR (qRT-PCR) is an alternative method for ER and PR testing and while concordance with IHC has been reported, additional testing is merited. This study compares ER, PR and HER2 status determined by IHC and qRT-PCR. Methods: FFPE tissues of ER(+) tumors collected and tested at the Blumenthal Cancer Center were studied. Expression levels of ESR1, PGR, and ERBB2 were determined by a multiplex qRT-PCR TaqMan assay and by the Oncotype Dx assay. Pre-established cutpoints were used to determine positivity. Only samples with qRT-PCR and corresponding IHC data were used, resulting in 144 ER, 128 PR, and 107 HER2 comparisons. ESR1 and PGR expression levels determined by the two qRT-PCR assays were also compared. Results: Of the144 IHC ER(+) samples, 142 were positive and 2 were negative by both qRT-PCR assays. All 120 IHC PR(+) samples were positive by both qRT-PCR assays. Of the 8 IHC PR(-) samples, 5 were negative and 3 were positive by both qRT-PCR assays. Of the 107 IHC HER2(-) samples, all but 2 were negative by qRT-PCR. One sample was positive by both qRT-PCR assays; one was positive by Oncotype Dx only. The expression levels determined by the qRT-PCR assays showed good correlation for ESR1 (r=0.85) and PGR (r=0.9). Conclusions: Concordance between qRT-PCR and IHC was 98.6% for ER and 97.7% for PR. Except for a single HER2 determination, there was 100% concordance between the two qRT-PCR assays. The discordant sample was HER2 (+) by multiplex qRT-PCR and “equivocal” by Oncotype Dx. Excellent correlation was also observed in the mRNA expression levels. The absence of HER2(+) and ER(-) samples are limitations of this study. These results suggest that qRT-PCR is a promising alternative method to IHC for determining hormone receptor status. Additional testing of samples with endocrine therapy outcomes including ER(-) samples would be beneficial.

Different patterns of expression and of IL-10 modulation of inflammatory mediators from macrophages of Lyme disease-resistant and -susceptible mice.

C57BL/6J (C57) mice develop mild arthritis (Lyme disease-resistant) whereas C3H/HeN (C3H) mice develop severe arthritis (Lyme disease-susceptible) after infection with the spirochete Borrelia burgdorferi. We hypothesized that susceptibility and resistance to Lyme disease, as modeled in mice, is associated with early induction and regulation of inflammatory mediators by innate immune cells after their exposure to live B. burgdorferi spirochetes. Here, we employed multiplex ELISA and qRT-PCR to investigate quantitative differences in the levels of cytokines and chemokines produced by bone marrow-derived macrophages from C57 and C3H mice after these cells were exposed ex vivo to live spirochetes or spirochetal lipoprotein. Upon stimulation, the production of both cytokines and chemokines was up-regulated in macrophages from both mouse strains. Interestingly, however, our results uncovered two distinct patterns of spirochete- and lipoprotein-inducible inflammatory mediators displayed by mouse macrophages, such that the magnitude of the chemokine up-regulation was larger in C57 cells than it was in C3H cells, for most chemokines. Conversely, cytokine up-regulation was more intense in C3H cells. Gene transcript analyses showed that the displayed patterns of inflammatory mediators were associated with a TLR2/TLR1 transcript imbalance: C3H macrophages expressed higher TLR2 transcript levels as compared to those expressed by C57 macrophages. Exogenous IL-10 dampened production of inflammatory mediators, especially those elicited by lipoprotein stimulation. Neutralization of endogenously produced IL-10 increased production of inflammatory mediators, notably by macrophages of C57 mice, which also displayed more IL-10 than C3H macrophages. The distinct patterns of pro-inflammatory mediator production, along with TLR2/TLR1 expression, and regulation in macrophages from Lyme disease-resistant and -susceptible mice suggests itself as a blueprint to further investigate differential pathogenesis of Lyme disease.

Detection and Typing of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus by Multiplex Real-Time RT-PCR

Porcine reproductive and respiratory syndrome (PRRS) causes economic losses in the pig industry worldwide, and PRRS viruses (PRRSV) are classified into the two distinct genotypes “North American (NA, type 2)” and “European (EU, type 1)”. In 2006, a highly pathogenic NA strain of PRRSV (HP-PRRSV), characterized by high fever as well as high morbidity and mortality, emerged in swine farms in China. Therefore, a real-time reverse transcription polymerase chain reaction (RT-qPCR) assay specific for HP-PRRSV was developed and combined with type 1- and type 2-specific RT-qPCR systems. Furthermore, an internal control, based on a heterologous RNA, was successfully introduced. This final multiplex PRRSV RT-qPCR, detecting and typing PRRSV, had an analytical sensitivity of less than 200 copies per µl for the type 1-assay and 20 copies per µl for the type 2- and HP assays and a high diagnostic sensitivity. A panel of reference strains and field isolates was reliably detected and samples from an animal trial with a Chinese HP-PRRS strain were used for test validation. The new multiplex PRRSV RT-qPCR system allows for the first time the highly sensitive detection and rapid differentiation of PRRSV of both genotypes as well as the direct detection of HP-PRRSV.

Neuropeptide Y2 Receptor (NPY2R) Expression in Saliva Predicts Feeding Immaturity in the Premature Neonate

BackgroundThe current practice in newborn medicine is to subjectively assess when a premature infant is ready to feed by mouth. When the assessment is inaccurate, the resulting feeding morbidities may be significant, resulting in long-term health consequences and millions of health care dollars annually. We hypothesized that the developmental maturation of hypothalamic regulation of feeding behavior is a predictor of successful oral feeding in the premature infant. To test this hypothesis, we analyzed the gene expression of neuropeptide Y2 receptor (NPY2R), a known hypothalamic regulator of feeding behavior, in neonatal saliva to determine its role as a biomarker in predicting oral feeding success in the neonate.Methodology/Principal FindingsSalivary samples (n = 116), were prospectively collected from 63 preterm and 13 term neonates (post-conceptual age (PCA) 26 4/7 to 41 4/7 weeks) from five predefined feeding stages. Expression of NPY2R in neonatal saliva was determined by multiplex RT-qPCR amplification. Expression results were retrospectively correlated with feeding status at time of sample collection. Statistical analysis revealed that expression of NPY2R had a 95% positive predictive value for feeding immaturity. NPY2R expression statistically significantly decreased with advancing PCA (Wilcoxon test p value<0.01), and was associated with feeding status (chi square p value = 0.013).Conclusions/SignificanceDevelopmental maturation of hypothalamic regulation of feeding behavior is an essential component of oral feeding success in the newborn. NPY2R expression in neonatal saliva is predictive of an immature feeding pattern. It is a clinically relevant biomarker that may be monitored in saliva to improve clinical care and reduce significant feeding-associated morbidities that affect the premature neonate.

Fast‐mode duplex qPCR for BCR‐ABL1 molecular monitoring: Innovation, automation, and harmonization

Reverse transcription quantitative polymerase chain reaction (RTqPCR)is currently the most sensitive tool available for the routine monitoring of disease level in patients undergoing treatment for BCRABL1 associated malignancies. Considerable effort has been invested at both the local and international levels to standardise the methodology and reporting criteria used to assess this critical metric. In an effort to accommodate the demands of increasing sample throughput and greater standardization, we adapted the current best-practice guidelines to encompass automation platforms and improved multiplex RT-qPCR technology.

Abstract LB-306: Multiplex RT-qPCR study on the expression of stem cell markers CD24, CD44, and ALDH1 in circulating tumor cells of breast cancer patients

Abstract Background: Molecular characterization of Circulating Tumor Cells (CTC) can give important information on their suspected connection to cancer stem cells. In breast cancer, putative cancer stem cells have been identified to have a CD44+CD24-/low and ALDH1+ cell phenotype. Patients and methods: We developed a tetraplex quantitative RT-qPCR assay for CD24, CD44, ALDH1 and HPRT based on the in-silico design of primers and probes with different fluorescence emission spectra. Specificity and sensitivity experiments were performed using the SKBR-3 cancer cell line. We isolated CTC from peripheral blood according to our previously described methodology (Strati et al, BMC cancer 2011) from 40 patients with early breast cancer before the administration of adjuvant chemotherapy, 23 patients with verified metastasis and 18 female healthy volunteers. We evaluated all our data with respect to CD24, CD44, ALDH1 expression of healthy volunteers using the 2βΔΔCt method. Results: In 23 patients with verified metastasis 9.0% were found to have CD44+CD24-/low phenotype and 13% ALDH1high/CD24-/low phenotype. In 40 operable breast cancer patients 8 patients (20.0%) were found to have CD44+CD24-/low phenotype and 12 patients (30.0%) were found to have ALDH1high/CD24-/low phenotype. CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+, and mammaglobin gene transcripts were also quantified in the same samples. Discussion: Molecular characterization of CTC has shown that in breast cancer patients CTCs are partly characterized by a putative stem cell phenotype. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome of these patients is known. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-306. doi:1538-7445.AM2012-LB-306

Single-cell analysis of murine long-term hematopoietic stem cells reveals distinct patterns of gene expression during fetal migration.

BackgroundLong-term hematopoietic stem cells (LT-HSCs) migrate from the fetal liver (FL) to the fetal bone marrow (FBM) during development. Various adhesion and chemotactic receptor genes have been implicated in the migration of adult LT-HSCs. However, their role in the migration of fetal LT-HSCs is not clearly understood due, in part, to the rare number of these cells in fetal tissues, which preclude classical gene expression analysis. The aim of this study is to characterize the expression of migration related genes in fetal LT-HSC across different anatomical locations during development.Methodology/Principal FindingsWe isolated fetal LT-HSC from different developmental stages, as well as different anatomical locations, and performed single-cell multiplex RT-qPCR and flow cytometry analysis of eight molecules involved in adult LT-HSC migration. Our results show that the gene expression of the chemokine receptor Cxcr4 in LT-HSC varies across developmental microenvironments and times, while the cadherin Cdh2 (Ncad) and the calcium receptor Casr show higher gene expression and variability only in FBM at 17.5 days post coitum (dpc). The cadherin Cdh5 (Vecad) maintains high expression variability only during fetal development, while the integrin subunit Itga5 (α5) increases its variability after 14.5 dpc. The integrin subunits Itga4 (α4) and Itgal (Lfa1), as well as the selectin ligand Selplg (Psgl1), did not show differences in their expression in single LT-HSCs irrespective of the developmental times or anatomical microenvironments studied.Conclusions/SignificanceOur data demonstrate that the expression pattern of phenotypically identical, single LT-HSCs fluctuates as a function of developmental stage and anatomical microenvironment. This is the first exhaustive gene expression comparison of migration-related molecules in fetal tissues across developmental times, enhancing the understanding of LT-HSC migration fate decisions during development.

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.