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AbstractThe amount of available DNA is often a limiting factor in pursuing genetic analyses of large-scale population cohorts. An association between higher DNA yield from blood and several phenotypes associated with inflammatory states has recently been demonstrated, suggesting that exclusion of samples with very low DNA yield may lead to biased results in statistical analyses. Whole genome amplification (WGA) could present a solution to the DNA concentration-dependent sample selection. The aim was to thoroughly assess WGA for samples with low DNA yield, using the multiply-primed rolling circle amplification method. Fifty-nine samples were selected with the lowest DNA yield (less than 7.5?g) among 799 samples obtained for one population cohort. The genotypes obtained from two replicate WGA samples and the original genomic DNA were compared by typing 24 single nucleotide polymorphisms (SNPs). Multiple genotype discrepancies were identified for 13 of the 59 samples. The largest portion of discrepancies was due to allele dropout in heterozygous genotypes in WGA samples. Pooling the WGA DNA replicates prior to genotyping markedly improved genotyping reproducibility for the samples, with only 7 discrepancies identified in 4 samples. The nature of discrepancies was mostly homozygote genotypes in the genomic DNA and heterozygote genotypes in the WGA sample, suggesting possible allele dropout in the genomic DNA sample due to very low amounts of DNA template. Thus, WGA is applicable for low DNA yield samples, especially if using pooled WGA samples. A higher rate of genotyping errors requires that increased attention be paid to genotyping quality control, and caution when interpreting results.

This study describes a practical system that allows high-throughput genotyping of single nucleotide polymorphisms (SNPs) and detection of mutations by allele-specific extension on primer arrays. The method relies on the sequence-specific extension of two immobilized allele-specific primers that differ at their 3'-nucleotide defining the alleles, by a reverse transcriptase (RT) enzyme at optimized reaction conditions. We show the potential of this simple one-step procedure performed on spotted primer arrays of low redundancy by generating over 8000 genotypes for 40 mutations or SNPs. The genotypes formed three easily identifiable clusters and all known genotypes were assigned correctly. Higher degrees of multiplexing will be possible with this system as the power of discrimination between genotypes remained unaltered in the presence of over 100 amplicons in a single reaction. The enzyme-assisted reaction provides highly specific allele distinction, evidenced by its ability to detect minority sequence variants present in 5% of a sample at multiple sites. The assay format based on miniaturized reaction chambers at standard 384-well spacing on microscope slides carrying arrays with two primers per SNP for 80 samples results in low consumption of reagents and makes parallel analysis of a large number of samples convenient. In the assay one or two fluorescent nucleotide analogs are used as labels, and thus the genotyping results can be interpreted with presently available array scanners and software. The general accessibility, simple set-up, and the robust procedure of the array-based genotyping system described here will offer an easy way to increase the throughput of SNP typing in any molecular biology laboratory.

The vascular endothelial growth factor family (VEGF, VEGF-B, -C and -D) is the main regulator of angiogenesis and lymphangiogenesis via their receptors, Flt-1, KDR and Flt-4. The aim of the present study was to estimate the microvascular density and investigate the expression of VEGF, VEGF-C and its receptors Flt-1, KDR and Flt-4 in peripheral T-cell lymphomas (PTCL). Material and methods: Microvessel density (MVD) was determined after CD34 immunohistochemical (IH) staining of endothelial cells in pre-therapeutic lymph-node biopsies from 107 cases of PTCL (64 unspecified, 1 angiocentric, 10 angioimmunoblastic and 32 anaplastic large cell T/0). 44 of these cases (28 unspecified, 1 angiocentric, 4 angioimmunoblatic and 11 anaplastic large cell T/0) were investigated for expression of angiogenic molecules, VEGF, VEGF-C and their receptors by IH at protein level. Furthermore, VEGF and VEGF-C mRNA expression was detected by non-isotopic ′in situ′ hybridization. The angiogenic scores were correlated to pre-therapeutic clinical parameters, treatment response and survival. Results: Tumoral expression of VEGF, VEGF-C and of their receptors was detected in the majority of the PTCL biopsies investigated (VEGF mRNA: 73%, VEGF protein: 95%; VEGF-C mRNA: 79%; VEGF-C protein: 79%; Flt-1 and KDR: 100%; Flt-4: 26%). All biopsies contained VEGF, Flt-1 and KDR positive vessels and VEGF-C was also widely expressed by the endothelial cells (3 negative cases), while endothelial Flt-4 expression was detected only in 43% of cases. High MVD scores correlated with an advanced clinical stadium (Ann Arbor) (p=0.047). A strong diffuse VEGF mRNA expression significantly correlated with a poorer overall survival (p=0.0015) as compared to focal or negative ISH signal. The predictive value of this parameter persisted at multivariate level independently of the International Prognostic Index (HR: 3.95, p=0.012, Cox regression). Conclusion: VEGF, VEGF-C and their receptors are expressed by both lymphoma and endothelial cells in the majority of PTCL cases. VEGF expression seems to have an adverse impact on survival. Larger studies of angiogenesis/lymphangiogenesis in PTCL are needed to clarify the biological role of these molecules and identify possible therapeutic targets.

Background: VEGF is the main regulator of tumour angiogenesis via its receptors, Flt-1 and KDR. It is expressed by tumour cells and normal stroma cells, and, beyond angiogenesis, it can stimulate tumour growth via autocrine and paracrine pathways. Material and methods: Pre-therapeutic lymph-node biopsies from 152 NHL cases were investigated. These included 62 cases of follicular lymphoma (FL), 20 of which had histologically verified transformation to aggressive lymphoma, 46 cases of diffuse large B-cell lymphoma (DLBCL) and 44 cases of peripheral T-cell lymphoma (PTCL). VEGF, Flt-1 and KDR were investigated at protein level by immunohistochemistry, and at mRNA level by non-isotopic’ in situ’ hybridisation. For the latter, a fluorescein-conjugated probe was obtained from pGEM3Zf plasmid containing 1–581 bp of human VEGF cDNA. The staining intensity was scored semiquantitatively (0–3) and subdivided in ‘focal’or ‘diffuse’depending on morphological evaluation. Expression of VEGF and its receptors was correlated to the morphologically estimated microvascular density (Chalkley method). Results: VEGF protein expression was detected in 88 % FL, 91% DLBCL and 95% PTCL. As expected, PTCL had a stronger and more diffuse staining pattern than FL and DLBCL. Flt-1 protein expression was detected in 95 % FL, 96% DLBCL and in virtually all cases of PTCL. Lymphoma cells expressing KDR were also seen in all biopsies. A diffuse staining pattern was more frequent in FL. VEGF mRNA was expressed in all subtypes at similar levels although DLBCL displayed a diffuse staining pattern more frequently than other histologies. Higher VEGF protein expression in lymphoma cells was correlated with higher microvascular density (p=0.005), while a negative correlation was observed between receptor expression (Flt-1 and KDR) and Chalkley count (p=0.01 and 0.003, respectively). Patients with FL, who had diffuse VEGF staining of lymphoma cells, had poorer overall survival than those with focal staining (p=0.04). Diffuse KDR staining was correlated with a higher risk of transformation (p=0.05). In fact, a remarkably high microvessel density was found in interfollicular as opposed intrafollicular areas and high interfollicular Chalkley count was correlated with histologic transformation to DLBCL (p=0.01). In ‘de novo’ DLBCL, high KDR expression was correlated with poor treatment response (p=0.03) and a grade 3 KDR intensity in lymphoma cells had a significant adverse impact on overall survival (p<0.001). In PTCL, a diffuse tissue distribution of VEGF mRNA correlated to poorer overall survival (p=0.0015). Flt-1 expression had no influence on outcome in the investigated lymphoma subtypes Conclusion: VEGF and its receptors are expressed by lymphoma cells in all investigated NHL subtypes. The staining pattern and intensity of VEGF and KDR differed in the three subtypes, while Flt-1 distribution was similar. High VEGF and KDR expression had adverse prognostic impact in DLBCL and PTCL.