No abstract

Thyroid cancer is the most prevalent type of endocrine malignancy. During the past decades its incidence has been increasing in many countries [1]. Papillary thyroid carcinoma (PTC) accounts for more than 80% of all thyroid cancers and for 95% of this increase [2]. About 70% of the newly diagnosed PTCs are small 852-8523, JapanAbstract. Molecular pathogenesis of papillary thyroid carcinoma (PTC) is largely associated with mutational changes in the BRAF, RAS family and RET genes. Our aim was to assess clinico-pathological and prognostic correlations of these PTCspecific gene alterations, with a particular emphasis on the BRAF mutation, in a group of 266 Serbian PTC patients, for the first time. The reference center-based retrospective cohort included 201 (75.6%) females and 65 (24.4%) males aged 48.0±16.1 years (8 -83 years old, range) diagnosed and treated for PTC during 1993-2008. Follow-up period was 53.1±41.6 months (7-187 months, range). BRAF and RAS mutations were determined by direct sequencing of genomic DNA. RET/ PTC rearrangements were analyzed by RT-PCR/Southern blotting. Genetic alterations were detected in 150/266 tumors (56.4%). One tumor displayed two genetic alterations. The BRAF V600E was found in 84/266 (31.6%) cases, RAS mutations in 11/266 (4.1%) and RET/PTC in 55/266 (20.7%; 42/266 (15.8%) RET/PTC1 and 13/266 (4.9%) RET/PTC3). On multivariate analysis BRAF V600E was associated with the classical papillary morphology (P = 0.05), the higher pT category (P = 0.05) and advanced clinical stage (P = 0.03). In a proportional hazard model, BRAF V600E did not appear to be an independent risk factor for the faster recurrence (P = 0.784). We conclude that under the extensive thyroid surgery and limited application of radioiodine ablation BRAF V600E may not be an indicator of poorer disease-free survival during the short to middle follow-up period. However, it has a potential to contribute to patients stratification into high-and low-risk groups.

MicroRNA-21 (miR-21) overexpression is characteristic for various types of tumors, but it is still unknown whether its expression levels differ between invasive and non-invasive breast carcinomas. The main goal of the study was to determine the difference in miR-21 expression among normal tissue, non-invasive, invasive with non-invasive component, and pure invasive breast cancer samples, to explain its potential role and significance in breast cancer invasiveness. The second goal was to propose miR-21 as molecular marker of breast cancer invasiveness and potential target for future anti-miR therapies for the prevention of invasion and metastasis. In order to reveal the role of miR-21 in breast cancer invasiveness, we measured miR-21 expression levels in 44 breast cancer and four normal samples by stem-loop real-time RT-PCR using TaqMan technology. Relative expression levels of miR-21 were significantly higher in invasive than in other groups (P=0.002) and significantly higher in invasive compared with invasive with non-invasive component group in histological (P=0.043) and nuclear grade 2 (P=0.036), estrogen-receptor-positive (ER+) (P=0.006), progesterone-receptor-positive (PR+) (P=0.008), ER+PR+ (P=0.007), and proliferation index (Ki-67)≤20% (P=0.036) tumors. Our findings suggest that miR-21 could be independent molecular marker of breast cancer invasiveness and potential target for future anti-miR therapies for the prevention of invasion and metastasis.

Although Pst I does not cut the circular mitochondrial genome of the rat, BamHI generates from this genome two unequal fragments of DNA. Each of these fragments was cloned in pBR322. Nuclear DNA was digested from rat liver singly or doubly with Pst I and BamHI, and it was demonstrated that nuclear DNA shared a common sequence with the larger mitochondrial DNA BamHI fragment. The cloned larger mitochondrial DNA fragment was further subdivided with HidMII into four pieces that were labeled and then used to probe the double-digested nuclear DNA. The hybridization data showed that the common sequence is less than 3 kilobase pairs long and lies within the part of the mitochondrial genome containing the D-loop and a portion of the rRNA genes. It therefore appears that, as in lower eukaryotes, there are shared sequences between the nuclear and mitochondrial genomes in mammals.

AIM:To investigate the significance of p 16 and O 6 -methylguanine-DNA methyltransferase (MGMT ) genes promoter hypermethylation and K-ras mutations on colorectal tumorigenesis and progression.METHODS: p 16 and MGMT methylation status was examined on 47 tumor samples, and K-ras mutational s t a t u s wa s e x a mine d o n 85 t umo r s a mple s . Fo r methylation analysis, a methylation specific PCR (MS-PCR) method was used.RESULTS: p 16 and MGMT promoter methylation was found in 51% (24/47) and 43% (20/47) of CRCs, respectively, and the K-ras mutation was found in 44% (37/85) of CRCs. Comethylation of p 16 and MGMT genes was significantly associated with lower aggressiveness of the disease within a two-year period of observation.Only 27% of patients with simultaneous p 16 and MGMT methylation showed the detectible occurrence of metastasis and/or death, compared to 67% of patients without double methylation or with no methylation (3/11 vs 22/33, P < 0.05, χ 2 -test). In addition, p 16 and MGMT comethylation showed a trend toward an association with longer survival in patients with CRCs (35.5 ± 6.0 mo vs 23.1 ± 3.2 mo, P = 0.072, Log-rank test). Progression of the disease within a two-year period was observed in 66% of patients carrying the K-ras mutation, compared to only 19% of patients with wild type K-ras (29/44 vs 7/37, P < 0.001, χ 2 -test). The presence of the K-ras mutation significantly correlated to shortened overall survival (20.0 ± 1.9 mo vs 37.0 ± 1.8 mo, P < 0.001, Logrank test). The comethylation of p 16 and MGMT genes was significantly associated with lower aggressiveness of the disease even when K-ras mutations were included in the analysis as an independent variable. CONCLUSION:Our data suggest that comethylation of promoters of p 16 and MGMT genes could have a prognostic value in patients with CRC. Specifically, concurrent methylation of both genes correlates with better prognosis.

Tamoxifen is a standard therapeutical treatment in patients with estrogen receptor positive breast carcinoma. However, less than 50% of estrogen receptor positive breast cancers do not respond to tamoxifen treatment whereas 40% of tumors that initially respond to treatment develop resistance over time. The underlying mechanisms for tamoxifen resistance are probably multifactorial but remain largely unknown. The primary aim of this study was to investigate the impact of PTEN tumor suppressor gene on acquiring resistance to tamoxifen by analyzing loss of heterozygosity (LOH) and immunohystochemical expression of PTEN in 49 primary breast carcinomas of patients treated with tamoxifen as the only adjuvant therapy. The effect of PTEN inactivation on breast cancer progression and disease outcome was also analyzed. Reduced or completely lost PTEN expression was observed in 55.1% of samples, while 63.3% of samples displayed LOH of PTEN gene. Inactivation of PTEN immunoexpression significantly correlated with the PTEN loss of heterozygosity, suggesting LOH as the most important genetic mechanism for the reduction or complete loss of PTEN expression in primary breast carcinoma. Most importantly, LOH of PTEN and consequential reduction of its immunoexpression showed significant correlation with the recurrence of the disease. Besides, our study revealed that LOH of PTEN tumor suppressor was significantly associated with shorter disease free survival, breast cancer specific survival and overall survival. In summary, our results imply that LOH of PTEN could be used as a good prognostic characteristic for the outcome of breast cancer patients treated with tamoxifen.

Leukoplakias, clinically identifiable premalignant lesions, often precede oral squamous cell carcinoma (OSCC). Identification of leukoplakias that have the potential for transformation to malignancy is a key clinical problem. The aim of this study was to assess genomic instability, and to detect tumor-specific genomic alterations, in leukoplakias. Genomic instability was analyzed by comparing the DNA fingerprints of 32 leukoplakias with those of paired normal tissue. In addition, the mutational status of the p53 gene was analyzed using polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) and polymerase chain reaction-heteroduplex DNA (PCR-HET), and the mutations were subsequently confirmed by DNA sequencing. Moderate-to-significant genomic instability was detected in all leukoplakias analysed. Nine unique amplicons, present in leukoplakias but not in normal tissue, were retrieved and successfully characterized. The p53 gene was mutated in 40.6% of patients. Four patients with moderate instability and mutated p53 developed OSCC. The data obtained in this study support and concretize the thesis that premalignant lesions possess many of the alterations found in cancer before the development of a malignant phenotype. Inactivation or mutation of the p53 tumor-suppressor might be an early event contributing to genomic instability and increasing the risk of malignant transformation.

Specimens of malignant and normal female human breast tissues were analyzed after surgery by means of synchronous luminescence spectroscopy. Measurements were performed in the ranges of excitation wavelengths from 330 to 650 nm and synchronous wavelengths from 30 to 120 nm to obtain ordinary and first derivative three-dimensional total synchronous luminescence spectra (3d-TSLS) of each specimen. Arithmetic mean of these spectra has been calculated for normal and malignant specimens and analyzed to establish criteria for tissue differentiation. Spectral domain volumes (volumes below luminescence intensity surface) and mean spectral slopes have been calculated and also analyzed as tissue discrimination criteria. The obtained results are discussed in view of the possible relevance of synchronous luminescence spectroscopy in discrimination between normal and malignant breast tissue.