Detection of viruses infecting Lilium spp. by RT-PCR and Real-Time PCR

In order to enhance the Italian lily bulb production, a breeding program was carried out at CRA-VIV in Pescia (PT – Italy) during the last years. Asiatic hybrids (Lilium × elegans Thunb.), lily cultivars and other native species were involved in the program. The obtained lily lines, which presented interesting traits, were preserved in a collection. After years of vegetative propagation, some of the new selections showed symptoms referable to viral infections. Virus diseases represent some of the most dangerous threats of Lilium, so the application of fast and effective diagnostic techniques for early detection is very important. The aim of the present study, in the frame of a phytosanitary survey of the lily collection, is to investigate the presence and incidence of Cucumber mosaic virus (CMV), Lilium symptomless virus (LSV), Lily mottle virus (LMoV) and two tospoviruses (Impatiens necrotic spot virus, INSV, and Tomato spotted wilt virus, TSWV). Among the 60 samples object of this study, infections by LSV and CMV were frequently observed. Also LMoV was detected in a smaller amount of samples. All of the samples were negative to INSV and TSWV

Microsatellite instability in thyroid tumours and tumour-like lesions

Fifty-one thyroid tumours and tumour-like lesions were analysed for instability at ten dinucleotide microsatellite loci and at two coding mononucleotide repeats within the transforming growth factor β (TGF-β) type II receptor (TβRII) and insulin-like growth factor II (IGF-II) receptor (IGFIIR) genes respectively. Microsatellite instability (MI) was detected in 11 out of 51 cases (21.5%), including six (11.7%) with MI at one or two loci and five (9.8%) with Ml at three or more loci (RER+ phenotype). No mutations in the TβRII and IGFIIR repeats were observed. The overall frequency of MI did not significantly vary in relation to age, gender, benign versus malignant status and tumour size. However, widespread MI was significantly more frequent in follicular adenomas and carcinomas than in papillary and Hürthle cell tumours: three out of nine tumours of follicular type (33.3%) resulted in replication error positive (RER+), versus 1 out of 29 papillary carcinomas (3.4%, P = 0.01), and zero out of eight Hürthle cell neoplasms. Regional lymph node metastases were present in five MI-negative primary cancers and resulted in MI-positive in two cases. © 1999 Cancer Research Campaig

Identification of germline alterations of the mad homology 2 domain of SMAD3 and SMAD4 from the Ontario site of the breast cancer family registry (CFR)

Abstract Introduction A common feature of neoplastic cells is that mutations in SMADs can contribute to the loss of sensitivity to the anti-tumor effects of transforming growth factor-β (TGF-β). However, germline mutation analysis of SMAD3 and SMAD4, the principle substrates of the TGF-β signaling pathway, has not yet been conducted in breast cancer. Thus, it is currently unknown whether germline SMAD3 and SMAD4 mutations are involved in breast cancer predisposition. Methods We performed mutation analysis of the highly conserved mad-homology 2 (MH2) domains for both genes in genomic DNA from 408 non-BRCA1/BRCA2 breast cancer cases and 710 population controls recruited by the Ontario site of the breast cancer family registry (CFR) using denaturing high-performance liquid chromatography (DHPLC) and direct DNA sequencing. The results were interpreted in several ways. First, we adapted nucleotide diversity analysis to quantitatively assess whether the frequency of alterations differ between the two genes. Next, in silico tools were used to predict variants' effect on domain function and mRNA splicing. Finally, 37 cases or controls harboring alterations were tested for aberrant splicing using reverse-transcription polymerase chain reaction (PCR) and real-time PCR statistical comparison of germline expressions by non-parametric Mann-Whitney test of independent samples. Results We identified 27 variants including 2 novel SMAD4 coding variants c.1350G > A (p.Gln450Gln), and c.1701A > G (p.Ile525Val). There were no inactivating mutations even though c.1350G > A was predicted to affect exonic splicing enhancers. However, several additional findings were of note: 1) nucleotide diversity estimate for SMAD3 but not SMAD4 indicated that coding variants of the MH2 domain were more infrequent than expected; 2) in breast cancer cases SMAD3 was significantly over-expressed relative to controls (P A was associated with elevated germline expression (> 5-fold); 3) separate analysis using tissue expression data showed statistically significant over-expression of SMAD3 and SMAD4 in breast carcinomas. Conclusions This study shows that inactivating germline alterations in SMAD3 and SMAD4 are rare, suggesting a limited role in driving tumorigenesis. Nevertheless, aberrant germline expressions of SMAD3 and SMAD4 may be more common in breast cancer than previously suspected and offer novel insight into their roles in predisposition and/or progression of breast cancer

The Loss of the p53 Activator HIPK2 Is Responsible for Galectin-3 Overexpression in Well Differentiated Thyroid Carcinomas

Background: Galectin-3 (Gal-3) is an anti-apoptotic molecule involved in thyroid cells transformation. It is specifically overexpressed in thyroid tumour cells and is currently used as a preoperative diagnostic marker of thyroid malignancy. Gal-3 expression is downregulated by wt-p53 at the transcriptional level. In well-differentiated thyroid carcinomas (WDTCs) there is an unexplained paradoxical concomitant expression of Gal-3 and wt-p53. HIPK2 is a co-regulator of different transcription factors, and modulates basic cellular processes mainly through the activation of wt-p53. Since we demonstrated that HIPK2 is involved in p53-mediated Gal-3 downregulation, we asked whether HIPK2 deficiency might be responsible for such paradoxical Gal-3 overexpression in WDTC. Methodology/Principal Findings: We analyzed HIPK2 protein and mRNA levels, as well as loss of heterozygosity (LOH) at the HIPK2 locus (7q32-34), in thyroid tissue samples. HIPK2 protein levels were high in all follicular hyperplasias (FHs) analyzed. Conversely, HIPK2 was undetectable in 91.7% of papillary thyroid carcinomas (PTCs) and in 60.0% of follicular thyroid carcinomas (FTCs). HIPK2 mRNA levels were upregulated in FH compared to normal thyroid tissue (NTT), while PTC showed mean HIPK2 mRNA levels lower than FH and, in 61.5% of cases, also lower than NTT. We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas. To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression. HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation. Conclusions/Significance: Our results indicate that HIPK2 expression and function are impaired in WDTCs, in particular in PTCs, and that this event explains Gal-3 overexpression typically observed in these types of tumours. Therefore, HIPK2 can be considered as a new tumour suppressor gene for thyroid cancers

Low VHL mRNA Expression is Associated with More Aggressive Tumor Features of Papillary Thyroid Carcinoma

Alterations of the von Hippel-Lindau (VHL) tumor suppressor gene can cause different hereditary tumors associated with VHL syndrome, but the potential role of the VHL gene in papillary thyroid carcinoma (PTC) has not been characterized. This study set out to investigate the relationship of VHL expression level with clinicopathological features of PTC in an ethnically and geographically homogenous group of 264 patients from Serbia, for the first time. Multivariate logistic regression analysis showed a strong correlation between low level of VHL expression and advanced clinical stage (OR55.78, 95% CI 3.17-10.53, P<0.0001), classical papillary morphology of the tumor (OR52.92, 95% CI 1.33-6.44, P=50.008) and multifocality (OR51.96, 95% CI 1.06-3.62, P=50.031). In disease-free survival analysis, low VHL expression had marginal significance (P=50.0502 by the log-rank test) but did not appear to be an independent predictor of the risk for chance of faster recurrence in a proportion hazards model. No somatic mutations or evidence of VHL downregulation via promoter hypermethylation in PTC were found. The results indicate that the decrease of VHL expression associates with tumor progression but the mechanism of downregulation remains to be elucidated

Peripheral zone prostate cancer. Pre-treatment evaluation with MR and 3D (1)H MR spectroscopic imaging: correlation with pathologic findings

The purpose of this study was to retrospectively characterize benign and malignant prostate peripheral zone tissue by using endorectal MRI and 3D ¹H MRS. Fifty-two men with untreated biopsy-proven prostate cancer underwent combined endorectal MRI and MRSI. Whole-mount step-section histopathologic analysis constituted the reference standard. Biopsy correctly detected 74 locations; MRI correctly detected 72 locations; MRS correctly detected 72 locations; MRI + MRS correctly detected 78 locations. Cohen's test showed that biopsy had a lower degree of agreement with histology than MRI + MRS combined. The ratio of [(Cho + Cr)]/Cit correlates with the pathologic Gleason score. The addition of 3D¹H MRSI to MRI can improve diagnosis of prostate cancer contributing indirectly to improve local staging. In addition, the correlation between metabolic 3D¹H MRSI data with pathological Gleason grade may offer a non-invasive means to better predict prostate cancer aggressiveness