Maria Pla

A Specific Real-Time Quantitative PCR Detection System for Event MON810 in Maize YieldGard® Based on the 3′-Transgene Integration Sequence

The increasing presence of transgenic plant derivatives in a wide range of animal and human consumables has provoked in western Europe a strong demand for appropriate detection methods to evaluate the existence of transgenic elements. Among the different techniques currently used, the real-time quantitative PCR is a powerful technology well adapted to the mandatory labeling requirements in the European Union (EU). The use of transgene flanking genomic sequences has recently been suggested as a means to avoid ambiguous results both in qualitative and quantitative PCR-based technologies. In this study we report the identification of genomic sequences adjacent to the 3′-integration site of event MON810 in transgenic maize. This genetically modified crop contains transgene sequences leading to ectopic expression of a synthetic CryIA(b) endotoxin which confers resistance to lepidopteran insects especially against the European corn borer. The characterization of the genome–transgene junction sequences by means of TAIL-PCR has facilitated the design of a specific, sensitive and accurate quantification method based on TaqMan chemistry. Cloning of event MON810 3′-junction region has also allowed to compare the suitability of plasmid target sequences versus genomic DNA obtained from certified reference materials (CRMs), to prepare standard calibration curves for quantification.

A microarray-based detection system for genetically modified (GM) food ingredients

A multiplex DNA microarray chip was developed for simultaneous identification of nine genetically modified organisms (GMOs), five plant species and three GMO screening elements, i.e. the 35S promoter, the nos terminator and the nptII gene. The chips also include several controls, such as that for the possible presence of CaMV. The on-chip detection was performed directly with PCR amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required and on the number of primers present per amplification tube. The targets were biotin labelled and the arrays were detected using a colorimetric methodology. Specificity was provided by specific capture probes designed for each GMO and for the common screening elements. The sensitivity of the assay was tested by experiments carried out in five different laboratories. The limit of detection was lower than 0.3% GMO for all tests and in general around 0.1% for most GMOs. The chip detection system complies with the requirements of current EU regulations and other countries where thresholds are established for the labelling of GMO.

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

Development of real-time PCR systems based on SYBR® Green I, Amplifluor and TaqMan® technologies for specific quantitative detection of the transgenic maize event GA21

Abstract Maize GA21 line has integrated several tandemly repeated copies of the r-act 5-enol-pyruvylshikimate-3-phosphate synthase construct used for plant transformation. We were able to amplify a nucleotide sequence corresponding to the polylinker plasmid vector flanked by the r-act promoter and nopaline synthase 3′-terminator. A method for specific detection and quantification of Roundup Ready ® transgenic maize line GA21 DNA using conventional and real-time PCR and based on this transgenic sequence is described. GA21 specific primers and probe were designed targeting the vector–promoter junction region and amplifying a 72-bp DNA fragment. Quantification methods were optimized through three different real-time PCR chemistries, i.e. SYBR ® Green I, Amplifluor™ and TaqMan ® . All three methods proved to be specific, highly sensitive and reliable for both identification and quantification of GA21 DNA. Plasmid pGAivr containing single copies of the GA21 and invertase amplicons was constructed for use as external standard in calibration curves. Using pGAivr , a TaqMan ® based real-time PCR assay was optimized in duplex format targeting the maize species-specific ivr1 gene and the GA21 junction region. The detection limit of the method was 0.01% GA21, which is far below the established threshold for accidental presence of genetically modified organisms (GMO), this method therefore being suitable for use in routine GMO analysis.

Avoiding Axillary Treatment in Sentinel Lymph Node Micrometastases of Breast Cancer: A Prospective Analysis of Axillary or Distant Recurrence

BackgroundThe need for axillary lymph node dissection (ALND) in breast cancer patients with sentinel lymph node (SLN) micrometastases remains controversial. The aims of the study were to evaluate the locoregional failure and outcome of breast cancer patients with sentinel node micrometastases who did not undergo completion ALND.MethodsBetween November 2000 and December 2006, SLN biopsy was successfully performed in 1178 patients with invasive breast carcinoma. Only patients with macrometastasis (>2xa0mm) underwent ALND, while patients with negative SLN or micrometastases did not undergo further treatment of the axilla, by either surgery or radiotherapy. Regarding adjuvant therapy decision, patients with SLN-micrometastases (pN1mi) were considered as node-positive patients.ResultsOf 1,178 patients, 59 (5%) had micrometastases. Of those with micrometastases, 14 (24%) underwent ALND because the intraoperative study of the SLN yielded a positive result. With a median follow-up of 60 (range, 8–94) months, none of the patients with SLN micrometastases in whom ALND was omitted developed an axillary recurrence, while one patient in whom ALND was performed developed infraclavicular lymph node recurrence. One patient, who declined postoperative breast irradiation, developed breast recurrence and distant metastasis.ConclusionsBreast cancer patients with SLN micrometastases in whom ALND was omitted had a very low locoregional failure rate. This study supports the theory that ALND might be avoided in these patients, providing that adjuvant systemic treatment equal to treatment provided to treat node-positive disease is administered. However, longer follow-up and results of additional prospective studies are needed.

Patients with Breast Cancer and Negative Sentinel Lymph Node Biopsy without Additional Axillary Lymph Node Dissection: A Follow-Up Study of up to 5 Years

Objective: To analyze the rate of axillary recurrences and survival in patients operated on for breast cancer who had not undergone an axillary lymph node dissection (ALND) because of a negative sentinel node biopsy. Methods: The study includes 97 patients operated on for breast cancer and selective node biopsy from June 2000 to December 2001 who had a negative sentinel node biopsy and did not undergo ALND. Mean age was 58.2 years (55.9–60.5). Follow-up was done up to 5 years. After surgery all patients underwent clinical examination. Complementary treatment depended on the hospital protocol. Rate of axillary recurrences, presence of distant metastases and survival (Kaplan-Meier method) were studied. Results: After a median follow-up of 4.1 years (2.18–5.25), only 2/95 patients (2.1%) developed distant metastases. Four patients died but only the death of the patient who presented multiple metastases was related to the primary breast cancer (1%). The 5-year overall survival rate was 96%. Conclusions: (1) Only 1/95 patients studied developed nodal extra-axillary recurrence together with distant metastases. (2) The results obtained support the selective sentinel node biopsy as an accurate technique in the axillary stratification of patients with breast cancer, offering in the cases of negative sentinel node biopsy a safe axillary control after a 5-year follow-up.

Trends in the surgical procedures of women with incident breast cancer in Catalonia, Spain, over a 7-year period (2005–2011)

BackgroundBreast cancer (BC) is the most frequent cancer in women, accounting for 28% of all tumors among women in Catalonia (Spain). Mastectomy has been replaced over time by breast-conserving surgery (BCS) although not as rapidly as might be expected. The aim of this study was to assess the evolution of surgical procedures in incident BC cases in Catalonia between 2005 and 2011, and to analyze variations based on patient and hospital characteristics.MethodsWe processed data from the Catalonian Health Service’s Acute Hospital Discharge database (HDD) using ASEDAT software (Analysis, Selection and Extraction of Tumor Data) to identify all invasive BC incident cases according to the codes 174.0-174.9 of the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) that were attended for the one-year periods in 2005, 2008 and 2011. Patients were classified according to surgical procedures (BCS vs mastectomy, and immediate vs delayed reconstruction), and results were compared among periods according to age, stage, comorbidity and hospital level.ResultsBC surgical procedures were performed in more than 80% of patients. Surgical cases showed a significant increasing trend in the proportion of women aged 50–69 years, more advanced disease stages, higher comorbidity and they were attended in hospitals of less complexity level throughout the study period. Similar pattern was found for patients treated with BCS, which increased significantly from 67.9% in 2005 to 74.0% in 2011.Simple lymph node removal increased significantly (from 48.8% to 71.4% and from 63.6% to 67.8% for 2005 and 2011 in conservative and radical surgery, respectively). A slightly increase in the proportion of mastectomized young women (from 28% in 2005 to 34% in 2011) was detected, due to multiple factors. About 22% of women underwent post-mastectomy breast reconstruction, this being mostly immediate.ConclusionsThe use of HDD linked to the ASEDAT allowed us to evaluate BC surgical treatment in Catalonia. A consolidating increasing trend of BCS was observed in women aged 50–69 years, which corresponds with the pattern in most European countries. Among the mastectomized patients, immediate breast reconstructions have risen significantly over the period 2005–2011.

“Parallel” and “Antiparallel Tail‐Clamps” Increase the Efficiency of Triplex Formation with Structured DNA and RNA Targets

Sequence‐specific triple‐helix structures can be formed by parallel and antiparallel DNA clamps interacting with single‐stranded DNA or RNA targets. Single‐stranded nucleic acid molecules are known to adopt secondary structures that might interfere with intermolecular interactions. We demonstrate the correlation between a secondary structure involving the target—a stable stem predicted by in silico folding and experimentally confirmed by thermal stability and competition analyses—and an inhibitory effect on triplex formation. We overcame structural impediments by designing a new type of clamp: “tail‐clamps”. A combination of gel‐shift, kinetic analysis, UV thermal melting and thermodynamic techniques was used to demonstrate that tail‐clamps efficiently form triple helices with a structured target sequence. The performance of parallel and antiparallel tail‐clamps was compared: antiparallel tail‐clamps had higher binding efficiencies than parallel tail‐clamps both with structured DNA and RNA targets. In addition, the reported triplex‐stabilizing property of 8‐aminopurine residues was confirmed for tail‐clamps. Finally, we discuss the possible use of this improved triplex technology as a new tool for applications in molecular biology.

Efficacy and safety of concurrent trastuzumab plus weekly paclitaxel-FEC as primary therapy for HER2-positive breast cancer in everyday clinical practice.

One of the most efficacious primary therapies in HER2-positive breast cancer was published by the M.D. Anderson group in 2005. This randomized trial evaluated the addition of trastuzumab to a taxane–anthracycline based chemotherapy. Despite largely significant differences in pathological complete response (pCR) in the trastuzumab group (65 vs. 26xa0%) this regimen did not become a common standard due to toxicity concerns and its premature closure with a small sample size. In order to evaluate the efficacy and safety of this regimen in an off-trial setting we conducted a prospectively monitorized series of consecutive patients with early or locally advanced Her-2 positive breast cancer following the same treatment strategy. Stage II–IIIC HER2-positive breast cancer patients, including inflammatory disease, were treated with weekly-trastuzumab for 24xa0weeks administered concurrently with all primary chemotherapy containing paclitaxel (80xa0mg/m2) for 12xa0weeks and 4 cycles of FEC-75 (fluorouracil 500xa0mg/m2, epirubicine 75xa0mg/m2, and cyclophosphamide 500xa0mg/m2) followed by surgery. The objectives were efficacy, in terms of pCR in both the breast and lymph nodes, and safety, with close cardiac monitoring during and after treatment. From August 2004 to February 2009, 83 patients were included. Most patients (73.5xa0%) had node involvement and 13.2xa0% had inflammatory disease. Fifty-one patients (61.4xa0%) achieved a pCR in breast and axilla (95xa0% CI 50–72xa0%). HR-negative tumors were associated with higher pCR rate than HR-positive tumors (77 vs. 48xa0%, Pxa0=xa00.006). At a median follow-up of 50.2xa0months no patient developed symptomatic cardiac failure, and 9 patients (10.8xa0%) presented a transient asymptomatic decrease in left ventricular ejection fraction. Primary therapy with concurrent trastuzumab plus paclitaxel–FEC for HER2-positive breast cancer in everyday practice is highly effective and safe confirming the results observed in a randomized trial stopped prematurely.

Efficient sequence-specific purification of Listeria innocua mRNA species by triplex affinity capture with parallel tail-clamps

Parallel clamps can interact in a sequence‐specific manner with homopyrimidine DNA and RNA oligonucleotides to form triplexes. For longer nucleic acids, we have previously demonstrated the inhibitory effect of DNA‐target secondary structures on triplex formation. We further designed a modification of these molecules—that is, tail‐clamps formed by addition of a tail sequence to the parallel clamp—and proved efficient binding of the molecules with structured single‐stranded DNA targets. Here we explore the possible application of the tail‐clamp strategy for triplex formation with RNA targets, which are typically found as strongly folded single‐stranded molecules. Efficient and specific binding of a tail‐clamp designed to form a parallel triplex with Listeria innocua iap mRNA sequences has been verified by UV melting curves and triplex affinity capture techniques. Furthermore, we show for the first time the formation of stable complexes of mRNA with tail‐clamps not only under acidic but also under neutral and slightly basic pH conditions. These results signify a further step towards the possible applications of triplexes with mRNA molecules; research, analytical, and therapeutic uses can be envisaged. As an example, our tail‐clamp‐based triplex affinity capture assay allowed the specific capture and recovery of iap mRNA molecules from an L. innocua total RNA solution with 45u2009% yield.