Discriminating lymphomas and reactive lymphadenopathy in lymph node biopsies by gene expression profiling

Background

Diagnostic accuracy of lymphoma, a heterogeneous cancer, is essential for patient management. Several ancillary tests including immunophenotyping, and sometimes cytogenetics and PCR are required to aid histological diagnosis. In this proof of principle study, gene expression microarray was evaluated as a single platform test in the differential diagnosis of common lymphoma subtypes and reactive lymphadenopathy (RL) in lymph node biopsies.

Methods

116 lymph node biopsies diagnosed as RL, classical Hodgkin lymphoma (cHL), diffuse large B cell lymphoma (DLBCL) or follicular lymphoma (FL) were assayed by mRNA microarray. Three supervised classification strategies (global multi-class, local binary-class and global binary-class classifications) using diagonal linear discriminant analysis was performed on training sets of array data and the classification error rates calculated by leave one out cross-validation. The independent error rate was then evaluated by testing the identified gene classifiers on an independent (test) set of array data.

Results

The binary classifications provided prediction accuracies, between a subtype of interest and the remaining samples, of 88.5%, 82.8%, 82.8% and 80.0% for FL, cHL, DLBCL, and RL respectively. Identified gene classifiers include LIM domain only-2 (LMO2), Chemokine (C-C motif) ligand 22 (CCL22) and Cyclin-dependent kinase inhibitor-3 (CDK3) specifically for FL, cHL and DLBCL subtypes respectively.

Conclusions

This study highlights the ability of gene expression profiling to distinguish lymphoma from reactive conditions and classify the major subtypes of lymphoma in a diagnostic setting. A cost-effective single platform "mini-chip" assay could, in principle, be developed to aid the quick diagnosis of lymph node biopsies with the potential to incorporate other pathological entities into such an assay.

     

Genetic differentiation of two distinct chemotypes of Ferula communis (Apiaceae) in Sardinia (Italy)

The giant fennel, Ferula communis L. (Apiaceae) is a circum-Mediterranean species responsible of severe haemorrhagic intoxication of livestock. Electrophoretic analysis of 14 putative enzyme loci was carried out to assess genetic differentiation among poisonous and non-poisonous chemotypes of F. communis in Sardinia. Genetic structuring characterises all the five populations examined. However, differentiation was higher between poisonous and non-poisonous populations (P<0.00001), and was independent from geographic distances, suggesting a reduced gene flow between the two chemotypes.     

Gene expression profiling identifies activated growth factor signaling in poor prognosis (Luminal-B) estrogen receptor positive breast cancer

Background

For population based biorepositories to be of use, rigorous quality control and assurance must be maintained. We have designed and validated a panel of polymorphisms for individual sample identification consisting of 36 common polymorphisms that have been implicated in a wide range of diseases and an additional sex marker. This panel uniquely identifies our biorepository of approximately 20,000 samples and would continue to uniquely identify samples in biorepositories of over 100 million samples.

Methods

A panel of polymorphisms associated with at least one disease state in multiple populations was constructed using a cut-off of 0.20 or greater confirmed minor allele frequency in a European Caucasian population. The fingerprinting assay was tested using the MALDI-TOF mass spectrometry method of allele determination on a Sequenom platform with a panel of 28 Caucasian HapMap samples; the results were compared with known genotypes to ensure accuracy. The frequencies of the alleles were compared to the expected frequencies from dbSNP and any genotype that did not achieve Hardy Weinberg equilibrium was excluded from the final assay.

Results

The final assay consisted of the AMG sex marker and 36 medically relevant polymorphisms with representation on each chromosome, encompassing polymorphisms on both the Illumina 550K bead array and the Affymetrix 6.0 chip (with over a million polymorphisms) platform. The validated assay has a P(ID) of 6.132 × 10^-15 and a P_sib(ID) of 3.077 × 10^-8. This assay allows unique identification of our biorepository of 20,000 individuals as well and ensures that as we continue to recruit individuals they can be uniquely fingerprinted. In addition, diseases such as cancer, heart disease diabetes, obesity, and respiratory disease are well represented in the fingerprinting assay.

Conclusion

The polymorphisms in this panel are currently represented on a number of common genotyping platforms making QA/QC flexible enough to accommodate a large number of studies. In addition, this panel can serve as a resource for investigators who are interested in the effects of disease in a population, particularly for common diseases.     

Characterization of phenolic pellets for ESR dosimetry in photon beam radiotherapy

This work deals with the dosimetric features of a particular phenolic compound (IRGANOX 1076^®) for dosimetry of clinical photon beams by using electron spin resonance (ESR) spectroscopy. After the optimization of the ESR readout parameters (namely modulation amplitude and microwave power) to maximise the signal without excessive spectrum distortions, basic dosimetric properties of laboratory-made phenolic dosimeters in pellet form, such as reproducibility, dose–response, sensitivity, linearity and dose rate dependence were investigated. The dosimeters were tested by measuring the depth dose profile of a 6 MV photon beam. A satisfactory intra-batch reproducibility of the ESR signal of the manufactured dosimeters was obtained. The ESR signal proved to increase linearly with increasing dose in the investigated dose range 1–13 Gy. The presence of an intrinsic background signal limits the minimum detectable dose to a value of approximately 0.6 Gy. Reliable and accurate assessment of the dose was achieved, independently of the dose rate. Such characteristics, together with the fact that IRGANOX 1076^® is almost tissue-equivalent, and the stability of the ESR signal, make these dosimeters promising materials for ESR dosimetric applications in radiotherapy.     

Freeze-dried somatic cells direct embryonic development after nuclear transfer

The natural capacity of simple organisms to survive in a dehydrated state has long been exploited by man, with lyophylization the method of choice for the long term storage of bacterial and yeast cells. More recently, attempts have been made to apply this procedure to the long term storage of blood cells. However, despite significant progress, practical application in a clinical setting is still some way off. Conversely, to date there are no reports of attempts to lyophilize nucleated somatic cells for possible downstream applications. Here we demonstrate that lyophilised somatic cells stored for 3 years at room temperature are able to direct embryonic development following injection into enucleated oocytes. These remarkable results demonstrate that alternative systems for the long-term storage of cell lines are now possible, and open unprecedented opportunities in the fields of biomedicine and for conservation strategies.     

Nucleus transfer in mammals: how the oocyte cytoplasm modifies the transferred nucleus

Successful development of clones depends on the reprogramming of transferred nuclei in enucleated oocytes. Thus far, oocytes are the only cells that can convert nuclei, which are already differentiated, into undifferentiated stages resembling pronuclei in freshly fertilized zygotes and that can then complete development of the reconstructed embryos. However, we still don't know exactly how the enucleated oocyte (cytoplast) secures this reprogramming. Oocytes exhibit a number of cytoplasmic activities that may be involved reprogramming. We discuss how these activities may be involved in reprogramming of transferred nuclei.     

Donor-dependent developmental competence of oocytes from lambs subjected to repeated hormonal stimulation

An unpredictability of ovarian response still remains the major problem concerning ovine reproductive programs. The influence of several environmental, genetic, and ovarian cycle effects on oocyte/embryo yield from donor females has been previously reported. The present research has been designed to exclude aforementioned causes of variability, thus to verify embryogenic competence in homogenous groups of animals. For this purpose we used prepubertal ewes kept under identical conditions. Initially, we stimulated three groups of prepubertal ewes at various ages and used a number of gonadotropin treatments to assess differences in oocyte competence between individuals. The results revealed the repeatability of response within individual donor lambs throughout the study. Moreover, once the variability in both oocyte and embryo yield between homogenous groups of donors was revealed alongside the influence of age and type of gonadotropin treatment (P < 0.001), we investigated whether the individual donor effect persisted among genetically similar animals. Therefore, we compared oocyte and subsequent embryo output of sibling lambs derived from the most efficient donor. Here the genetic homogeneity of sisters kept under identical conditions substantially improved the uniformity of either follicular response or embryo production, suggesting that the genotype plays a primary role in establishing follicular recruitment and developmental capability of oocytes. This observation consents to predict the ovarian performance from a single ewe already in early prepuberty (i.e., to qualify the female to breeding programs).     

Enucleolation of porcine oocytes

Germinal vesicles (GVs) in immature mammalian oocytes contain prominent nucleoli whose role in the process of oocyte maturation is not fully understood. Here we report that the microsurgical removal of nucleoli from immature fully grown porcine oocytes permits germinal vesicle breakdown and chromosome condensation and the enucleolated oocytes mature up to the second metaphase. Interestingly, the enucleolation of growing oocytes which, although unable to mature, resulted in germinal vesicle breakdown and the formation of a cluster of condensed chromatin. These results indicate that the nucleolus in fully grown oocytes is dispensable at least for nuclear maturation. On the other hand, the results obtained in growing oocytes suggest the role of the nucleolus in the cell cycle regulation.     

Nicotine protects kidney from renal ischemia/reperfusion injury through the cholinergic anti-inflammatory pathway

Kidney ischemia/reperfusion injury (I/R) is characterized by renal dysfunction and tubular damages resulting from an early activation of innate immunity. Recently, nicotine administration has been shown to be a powerful inhibitor of a variety of innate immune responses, including LPS-induced toxaemia. This cholinergic anti-inflammatory pathway acts via the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). Herein, we tested the potential protective effect of nicotine administration in a mouse model of renal I/R injury induced by bilateral clamping of kidney arteries. Renal function, tubular damages and inflammatory response were compared between control animals and mice receiving nicotine at the time of ischemia. Nicotine pretreatment protected mice from renal dysfunction in a dose-dependent manner and through the alpha7nAChR, as attested by the absence of protection in alpha7nAChR-deficient mice. Additionally, nicotine significantly reduced tubular damages, prevented neutrophil infiltration and decreased productions of the CXC-chemokine KC, TNF-alpha and the proinflammatory high-mobility group box 1 protein. Reduced tubular damage in nicotine pre-treated mice was associated with a decrease in tubular cell apoptosis and proliferative response as attested by the reduction of caspase-3 and Ki67 positive cells, respectively. All together, these data highlight that nicotine exerts a protective anti-inflammatory effect during kidney I/R through the cholinergic alpha7nAChR pathway. In addition, this could provide an opportunity to overcome the effect of surgical cholinergic denervation during kidney transplantation.     

Cadmium removal from contaminated soil by thermally responsive elastin (ELPEC20) biopolymers

Cadmium contamination of soil is a major concern in the biosphere. Beyond the suite of available physico-chemical treatment methods, green and more efficient technologies are desired to reduce cadmium and other heavy metal contaminants to acceptable levels. Elastin-like polypeptides (ELP) composed of a polyhistidine domain (ELPH12) can be used as an environmentally benign chelating agent for ex situ soil washing. However, ELPH12 is relatively non-selective. A biopolymer with metal-binding domains that have stronger affinity, capacity, and selectivity would have distinct advantages. The aim of this work is to investigate the use of a new generation of ELP biopolymer, ELPEC20, containing synthetic phytochelatin (EC) as the metal-binding domain for ex situ soil washing. ELPEC20 was shown to bind cadmium more effectively and selectively than ELPH12. The binding constant of ELPEC20 is an order of magnitude higher and the binding capacity is fivefold higher than ELPH12. In contrast to ELPH12, no decrease in cadmium binding was observed in the presence of other competing metal ions. The improved selectivity and binding capacity provided by ELPEC20 were directly reflected in the enhanced cadmium extraction efficiency from contaminated soil. In batch washing studies up to 62% of the bound cadmium was removed by ELPEC20 while less than 12% was removed by ELPH12. Cadmium was removed not only from the exchangeable fraction but also the oxidizable fraction. The high-affinity binding sites of ELPEC20 also results in very rapid extraction with complete removal achieved within 1 h, suggesting that ELPEC20 could be used as part of a rapid (short retention time) technology with minimum possibility for the biodegradation of biopolymers.