Locally advanced rectal cancers with simultaneous occurrence of KRAS mutation and high VEGF expression show invasive characteristics.

In this study, we investigated the mutation status of KRAS gene in pretherapeutic and preoperative biopsies in 63 specimens of locally advanced rectal cancers in order to evaluate its potential predictive and/or prognostic role. Regions of interest of KRAS exon 2 were amplified and visualized on 2% agarose gel. Obtained PCR products were subjected to direct sequencing. KRAS mutations were detected in 35% of patients, 91% of which were located in codon 12 and 9% in codon 13. In general, KRAS mutation status did not affect the response to neoadjuvant chemoradiotherapy (CRT). However, patients harboring mutated KRAS gene, simultaneously with high vascular endothelial growth factor (VEGF) expression, exhibited a worse response to CRT (p=0.030), a more frequent appearance of local recurrences and distant metastasis (p=0.003), and shorter overall survival (p=0.001) compared to all others. On the contrary, patients with GGT>GCT KRAS mutation exhibited a significantly better response to CRT than those with any other type of KRAS mutation (p=0.017). Moreover, the presence of GGT>GCT mutation was associated with low VEGF and Ki67 expression (p=0.012 in both cases), parameters related to less aggressiveness of the disease. Our results suggest that KRAS mutation status could have some predictive and prognostic importance in rectal cancer when analyzed together with other parameters, such as VEGF and Ki67 expression. In addition, it seems that not only the presence but the type of KRAS mutation is important for examining its impact on CRT response.

Case with triple-negative breast cancer shows overexpression of both cFOS and TGF-ß1 in node-positive tissue.

We present herein a case report style article on a rare advanced triple-negative breast cancer (TNBC) patient with 6-month disease-free interval, and 10-month overall survival. Our results demonstrate that the poor clinical outcome of this patient was associated with pronounced, more than fivefold higher, overexpression of both cFOS and TGF-ß1 proteins in its metastatic nodal tissue extracts, when compared with the values of the two non-TNBC controls (with 'zero' disease-free interval and overall survival). This original observation suggests, for the first time, that both the cFOS and TGF-ß1 may be considered as a pair of biomarkers for an early assessment of poor prognosis for TNBC patients. The possible clinical implication of this observation is discussed.

Higher miR-21 expression in invasive breast carcinomas is associated with positive estrogen and progesterone receptor status in patients from Serbia.

MicroRNAs play essential role in breast carcinoma progression and invasion. Our principal goals were to assess clinicopathological and prognostic correlations of microRNA-21 (miR-21) expression levels in a group of 39 Serbian breast cancer patients with invasive lobular (ILC), ductal (IDC), or mixed (ILC-IDC) breast carcinomas and in order to discover the role of miR-21 in potential novel form of stratification of the patients with different estrogen receptor (ER) and progesterone receptor (PR) status. MiR-21 expression levels were measured by stem-loop real-time RT-PCR using TaqMan technology. ER, PR, human epidermal growth factor 2 receptor (Her-2), and proliferative index (Ki-67) were evaluated by immunohistochemistry. MiR-21 levels do not vary among ILC, IDC, and ILC-IDC subgroups. MiR-21 expression levels varied significantly in the age, tumor size, Ki-67, and different grade (p = 0.030, p = 0.036, p = 0.027 and p = 0.032, respectively) subgroups. ER+ and PR+ showed higher miR-21 levels than their negative receptor status paired groups ER- and PR- with p = 0.012 and p = 0.018, respectively. MiR-21 positively correlated with ER and PR status (p = 0.018, ρ = 0.379 and p = 0.034, ρ = 0.345, respectively). Our findings suggest that miR-21 emulates transitional form of expression and that the levels of expression might be useful for stratification of the patients with different receptor status with the purpose to seek for new therapy approaches especially for the patients with the lack of response to conventional endocrine therapy.

The difference in miR-21 expression levels between invasive and non-invasive breast cancers emphasizes its role in breast cancer invasion.

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.

p14(ARF) methylation is a common event in the pathogenesis and progression of myxoid and pleomorphic liposarcoma.

Liposarcoma represents the most frequent group of soft tissue sarcomas. The group can be divided into three different classes: (1) differentiated/undifferentiated (WDLPS/DDLPS), (2) myxoid/round cell (MLPS/RCLPS) and (3) pleomorphic liposarcoma (PLS). It has become apparent that p53-p14 and Rb-p16 pathways play important roles in the pathogenesis of various sarcoma types. Molecular studies of the genes involved in these two pathways showed wide variations between the liposarcoma subtypes or even within the same subtype. We sought to examine mutational status of p53 and methylation status of p16 (INK4a) /p14 (ARF) genes in primary and recurrent liposarcoma tumors. There were twelve myxoid (12/18, 66.7 %) and six pleomorphic liposarcoma (6/18, 33.3 %) samples. Immunohistochemical analysis revealed that p53 protein was overexpressed in 3/12 MLPS (25 %) and 6/6 PLS (100 %). Mutational analysis showed that 2/11 MLPS (18.2 %) and 2/6 PLS (33.3 %) contained mutated p53 gene. On the other hand, 3/18 samples (16.7 %) had methylated p16 promoter. However, the frequencies of the p14 (ARF) gene methylation were 83.3 % (10/12) and 50 % (3/6) in myxoid and pleomorphic group, respectively. Overall, 15 out of 18 (83.3 %) samples had either p53 gene mutation or methylated p14 (ARF) promoter. The results from the current study suggest significant impact of the p14 (ARF) gene methylation on the pathogenesis and progression of myxoid and to a lesser extent pleomorphic liposarcoma. Despite the limited number of samples, our study points to necessity of further investigation of p53-p14 and Rb-p16 pathways in liposarcoma.

Potential clinical significance of ERß ON promoter methylation in sporadic breast cancer.

The aim of the study was to assess how hypermethylation of the ON promoter of the estrogen receptor beta (ERß) gene affects its expression (at the mRNA and protein level) and to correlate these with some clinical and histopathological parameters. A total of 131 samples of frozen breast cancer tissue was analyzed. A custom-designed, two-step PCR method was used to measure the methylation index of the ERß gene ON promoter region. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to quantify mRNA of the ERß1 isoform, while ERß1 protein was determined using the Western blot method. There was a significant difference in the methylation index of the ERß gene ON promoter between the groups of patients with negative and positive axillary lymph node status (P = 0.03). In addition, the methylation index of the ON promoter was positively correlated with estrogen receptor alfa (ERα) protein levels (ρ = 0.31, P = 0.02). There was a significant difference in the methylation index of the ON promoter between the progesterone receptor (PR)-negative and PR-positive groups of patients (P = 0.01). ERß1 protein levels were negatively correlated with ERα protein (ρ = -0.27, P < 0.01). The methylation index of the ON promoter could be a more reliable additional parameter for prediction and/or prognosis in breast cancer than ERß1-mRNA and/or protein levels.

Prognostic significance of epigenetic inactivation of p16, p15, MGMT and DAPK genes in follicular lymphoma.

In this study, methylation-specific polymerase chain reaction was used to investigate the role and potential prognostic significance of the methylation status of p16, p15, MGMT and DAPK genes in 32 specimens of follicular lymphoma (FL). Hypermethylation of p15 gene was associated with lower hemoglobin level (P = 0.020) and MGMT/DAPK comethylation with relapsed disease (P = 0.018). Among all patients with FL, there was no significant difference in the overall survival between those with hypermethylated and unmethylated of any examined genes. Therefore, we analyzed methylation in the different groups according to FL International Prognostic Index (FLIPI) and tumor grade. In the high-risk group, patients with hypermethylated p16 gene had significant lower overall survival than those with unmethylated p16 (P = 0.006) and trend toward shorter failure-free survival (P = 0.068). In the same risk group, there was a trend toward longer overall survival for patients with hypermethylated MGMT gene, compared to those with unmethylated MGMT gene (P = 0.066). p15 methylation had impact on shorter overall survival in grade I group of patients (P = 0.013), and DAPK methylation tended to have impact on shorter failure-free survival in the whole examined group (P = 0.079). Our results suggest that promoter methylation of p16 and MGMT genes could have prognostic value when used in combination with the FLIPI and p15 methylation in combination with tumor grade. Concurrent methylation of MGMT and DAPK genes could be the marker of tumor chemoresistance and disease recurrence.

The impact of PTEN tumor suppressor gene on acquiring resistance to tamoxifen treatment in breast cancer patients.

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.

Concomitant aberrant methylation of p15 and MGMT genes in acute myeloid leukemia: association with a particular immunophenotype of blast cells.

In this study, methylation-specific polymerase chain reaction (MS-PCR) was used to define the methylation status of the target promoter sequences of p15 and MGMT genes in the group of 21 adult patients with acute myeloid leukemia (AML). The incidence of aberrant hypermethylation of p15 gene (71 %) was higher comparing to MGMT gene (33 %), whereas concomitant methylation of both genes had 24 % of the patients. Although the incidence of cytogenetic abnormalities between the groups with a different methylation status of p15 and/or MGMT genes was not significantly different, we observed general trend of clustering of abnormalities with adverse prognosis into groups with concomitant hypermethylation of both genes and only p15 gene. Also, we showed that AML patients with concomitant methylation of p15/MGMT genes had a higher proportion of leukemic blast cells characterized with specific expression of individual leukocyte surface antigens (CD117(+)/CD7(+)/CD34(+)/CD15(-)), indicating leukemic cells as early myeloid progenitors. Although we could not prove that hypermethylation of p15 and/or MGMT genes is predictive parameter for response to therapy and overall survival, we noticed that AML patients with comethylated p15/MGMT genes or methylated p15 gene exhibited a higher frequency of early death, lower frequency of complete remissions as well as a trend for shorter overall survival. Assessing of the methylation status of p15 and MGMT genes may allow stratification of patients with AML into distinct groups with potentially different prognosis.

Unilateral follicular variant of papillary thyroid carcinoma with unique KRAS mutation in struma ovarii in bilateral ovarian teratoma: a rare case report.

BACKGROUND: Struma ovarii (SO) is a rare form of ovarian mature teratoma in which thyroid tissue is the predominant element. Because of its rarity, the differential diagnosis between benign and malignant SO has not been clearly defined. It is believed that malignant transformation of SO has similar molecular features with and its prognosis corresponds to that of malignant tumors originating in the thyroid. CASE PRESENTATION: We report 35-year-old woman with bilateral ovarian cysts incidentally detected by ultrasound during the first trimester of pregnancy. Four months after delivery of a healthy child without complication she was admitted to the hospital for acute abdominal pain. Laparoscopic left adnexectomy was performed initially in a regional hospital; right cystectomy was done later in a specialized clinic. Intraoperative frozen section and a final pathology revealed that the cyst from the left ovary was composed of mature teratomatous elements, normal thyroid tissue (>50%) and a non-encapsulated focus of follicular variant of papillary thyroid carcinoma (PTC).Normal and cancerous thyroid tissues were tested for BRAF and RAS mutations by direct sequencing, and for RET/PTC rearrangements by RT-PCR/Southern blotting. A KRAS codon 12 mutation, the GGT → GTT transversion, corresponding to the Gly → Val amino acid change was identified in the absence of other genetic alterations commonly found in PTC. CONCLUSION: To the best of our knowledge, this is the first time this mutation is described in a papillary thyroid carcinoma arising in struma in the ovarii. This finding provides further evidence that even rare mutations specific for PTC may occur in such tumors. Molecular testing may be a useful adjunct to common differential diagnostic methods of thyroid malignancy in SO.

Different associations of estrogen receptor ß isoforms, ERß1 and ERß2, expression levels with tumor size and survival in early- and late-onset breast cancer.

BACKGROUND: In breast cancer, little is known about the consequences of co-expression of ERα with the second estrogen receptor, ERß, and its isoforms in light of their joint prognostic value. Previously reported correlations have been based mostly on independent ERα and ERß expression levels in breast tumors. PURPOSE: To address whether the expression ratio of ERα and ERß and its isoforms may be a more important parameter than their absolute levels, we analyzed relative mRNA expression ratios of ERß1 to ERß2 and ERα in 74 clinical samples of invasive breast cancer including 39 early-onset and 35 late-onset breast cancers. Expression levels were correlated with clinical and histopathological parameters and disease-free interval. RESULTS: A specific correlation of ERß1 expression levels with tumor size was detected in early-onset breast cancer patients and of ERß2 levels with tumor size in late-onset patients. Expression of both ERß isoforms inversely correlated with expression of the two estrogen regulated genes, progesterone receptor and pS2 in both groups. Higher levels of ERß2 than ERß1 isoform were associated with a better outcome in late-onset patients. CONCLUSIONS: Our results suggest that different isoforms of ERß may be involved in suppression of tumor growth in young and elder patients and may have different prognostic values.

Mutational and clinico-pathological analysis of papillary thyroid carcinoma in Serbia.

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 PTC-specific 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.

Application of supervised self-organizing maps in breast cancer diagnosis by total synchronous fluorescence spectroscopy.

Data from total synchronous fluorescence spectroscopy (TSFS) measurements of normal and malignant breast tissue samples are introduced in supervised self-organizing maps, a type of artificial neural network (ANN), to obtain diagnosis. Three spectral regions in both TSFS patterns and first-derivative TSFS patterns exhibited clear differences between normal and malignant tissue groups, and intensities measured from these regions served as inputs to neural networks. Histology findings are used as the gold standard to train self-organizing maps in a supervised way. Diagnostic accuracy of this procedure is evaluated with sample test groups for two cases, when the neural network uses TSFS data and when the neural network uses data from first-derivative TSFS. In the first case diagnostic sensitivity of 87.1% and specificity of 91.7% are found, while in the second case sensitivity of 100% and specificity of 94.4% are achieved.

Genomic instability and tumor-specific DNA alterations in oral leukoplakias.

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.

Concurrent quantitation of the A and D genotypes of hepatitis B virus.

Hepatitis B virus (HBV) infection is a global health problem associated with severe liver disorders. Viral load and HBV genotype affect the clinical outcome, guide antiviral therapy and provide long term prognosis for HBV infected patients. Various types of detection and quantitation assays are currently in use with a different effectiveness. The aim of this study was to develop a method that would provide simultaneous identification and quantitation of genotypes A and D in a single-tube reaction. Sera from infected patients were analyzed by a TaqMan based real time PCR. Optimized reagents were used for HBV DNA quantitation while the genotypes A and D were quantified separately by our design of the assay. Multiplex real time PCR was achieved and was specific for HBV genotypes A and D within a single-tube reaction. Simulation of mixed virus populations was identified reproducibly in vitro. Quantitation of these individual genotypes was exceptionally reliable, so much so that the sum of individual genotypes was equal to the total viral load determined in a separate reaction. Therefore, a straightforward, conceptually simple and reliable approach to issues involving HBV genotypes A and D is submitted. Identity and exact titer of these genotypes in the Caucasian population can now be determined easily.

Quantification of transforming growth factor beta 1 levels in metastatic axillary lymph node tissue extracts from breast cancer patients: a new specimen source.

OBJECTIVE: To use cytoplasmic tissue extract as a new specimen source to quantify transforming growth factor beta 1 (TGFbeta1) protein in metastatic axillary lymph node tissue (ALNT) of breast cancer (BC) patients and to confirm the feasibility of this approach in a prospective pilot study on a subgroup of patients with invasive BC. STUDY DESIGN: The 6 selected malignant and autologous nonmalignant pairs of ALNT were fractionated, under special preanalytical, nonaggressive/nondenaturing conditions, to obtain respective cytoplasmic extracts for TGFbeta1 detection by the Quantikine (R&D Systems Inc., Minneapolis, Minnesota, U.S.A.) enzyme-linked immunosorbent assay kit. RESULTS: The data indicated a highly significant (r = 0.973054) positive linear correlation between the TGFbeta1 concentration and total protein concentration in cytoplasmic extract of metastatic ALNT. The subsequent patients' pilot study, performed strictly before any clinicopathologic factors were accessible, revealed significantly (p < 0.01) elevated TGFbeta1 in malignant ALNT (median value: 1.05 ng/mg protein, range: 0.67-3.6 ng/mg protein, n = 6) vs. autologous nonmalignant ALNT controls (median value: 0.48 ng/mg protein, range: 0.29-0.90 ng/mg protein, n = 6). This elevation was correlated with the number of metastatic axillary lymph nodes with respect to the total and was consistent with an increase in size of tumor deposits in axillary lymph nodes. CONCLUSION: Our data provide for the first time suggestive evidence that the TGFbeta1 level in cytoplasmic extracts of metastatic ALNTs may be a promising biomarker of invasiveness for BC patients. Confirmatory, large-scale studies are needed to evaluate possible implications of this putative biomarker in BC diagnosis and treatment.

Expression of estrogen receptor beta wt isoform (ERbeta1) and ERbetaDelta5 splice variant mRNAs in sporadic breast cancer.

PURPOSE: In addition to Estrogen Receptor alpha (ERalpha) and Progesterone Receptor (PR), the Second Estrogen Receptor (ERbeta) appears to play an important role not only in estrogen signaling, but also in the pathogenesis of cancer in estrogen dependent tissues. The existence of various isoforms and splice variants of both ERs additionally complicates elucidation of their physiological role and involvement in the process of carcinogenesis. METHODS: In this study, the expression of ERbeta1 mRNA (wild type of beta receptor) and splice variant ERbetaDelta5 mRNA (which codes for truncated protein) was measured by the quantitative RT-PCR (q RT-PCR) in the 60 samples of Breast Cancer (BC) and correlated with ERalpha and PR protein levels and with clinical and histopathological parameters. RESULTS: We found the inverse correlation of ERbetaDelta5 mRNA expression with the levels of PR and ERalpha proteins in the group of postmenopausal patients; we also report the lower expression of ERbeta1 and ERbetaDelta5 mRNA in the larger tumors (>20 mm, T2, and T3) than in smaller ones (< or =20 mm, T1). The decrease of ERbetaDelta5 mRNA expression in larger tumors is found to arise from ER-positive breast carcinomas. In addition, the portion of tumors with concomitant high expression of both transcripts matches up the known percentage of tumors resistant to endocrine therapy in patients with different ER/PR status. CONCLUSIONS: As far as we know, this is the first study in which ERbetaDelta5 mRNA splice variant was quantified by real-time RT-PCR in the clinical samples of breast cancer tissue. Until now, the focus of clinical reports was the level of ERbeta1, ERbeta2, and ERbeta5 isoforms. The higher expression of ERbetaDelta5 mRNA is associated with the indicators of low biological aggressiveness of tumor (low tumor size within ER-positive status in our study) suggesting that the uncontrolled local tumor growth may occur as the expression of ERbetaDelta5 mRNA decreases in estrogen-dependent breast cancer.

Comethylation of p16 and MGMT genes in colorectal carcinoma: correlation with clinicopathological features and prognostic value.

AIM: To investigate the significance of p16 and O(6)-methylguanine-DNA methyltransferase (MGMT) genes promoter hypermethylation and K-ras mutations on colorectal tumorigenesis and progression. METHODS: p16 and MGMT methylation status was examined on 47 tumor samples, and K-ras mutational status was examined on 85 tumor samples. For methylation analysis, a methylation specific PCR (MS-PCR) method was used. RESULTS: p16 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 p16 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 p16 and MGMT methylation showed the detectable 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, chi(2)-test). In addition, p16 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, chi(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, Log-rank test). The comethylation of p16 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 p16 and MGMT genes could have a prognostic value in patients with CRC. Specifically, concurrent methylation of both genes correlates with better prognosis.

Identification of differentially expressed mRNA transcripts in drug-resistant versus parental human melanoma cell lines.

BACKGROUND: Malignant melanoma resistance to chemotherapy remains a major limitation to treatment. Our aim was to identify genes associated with drug resistance, in order to better understand the molecular events underlying the drug-resistant phenotype. MATERIALS AND METHODS: A human melanoma cell line and its drug-resistant variants obtained by selection with MNNG or 6-thioguanine were used. Alterations in gene expression were characterized by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Prominent mRNA fragments present in selected variants and not in the parental cells were identified and characterized by cloning and sequencing. Differential expression was confirmed by real-time RT-PCR. RESULTS: Three functionally distinct transcriptional products were demonstrated: the chaperonin subunit TCP 1-zeta-6A (CCT6A), the hyaluronan receptor CD44 and LPPR-2, the lipid phosphate phosphatase-related protein type-2. CONCLUSION: Genes with altered expression were identified in drug-resistant variants. The identified molecules may provide new insights into the molecular basis for melanoma resistance to chemotherapy.

Elevated plasma TGF-beta1 levels correlate with decreased survival of metastatic breast cancer patients.

BACKGROUND: The role of circulating TGF-beta(1) in prognosis of breast cancer (BC) was investigated with an intention to define TGF-beta(1)-dependent high risk and low risk subsets of patients. METHODS: Fifty three BC patients of all clinical stages and 37 healthy donors (HD) were analyzed for plasma TGF-beta(1) by the TbetaRII receptor-based Quantikine TGF-beta(1) ELISA kit. RESULTS: The plasma TGF-beta(1) level of Stage I/II disease (median: 0.94 ng/ml; n=10)) remained close to HD (median: 1.30 ng/ml; n=37; p>0.1). In contrast, Stage III/IV disease (median: 2.34 ng/ml; n=43) exhibited highly significant TGF-beta(1) elevation (p<0.001) relative to HD. Further analysis revealed that TGF-beta(1) increase was predominantly attributed to Stage IV, metastatic disease patients (Q3=4.23 ng/ml) rather than to the group Stage III/IV (Q3=3.58 ng/ml). Using the plasma TGF-beta(1) concentration of 3.00 ng/ml as the cut-off value, two subgroups of patients were formed. Overall 2-year survival of the first subgroup, having elevated plasma TGF-beta(1) (>3.00 ng/ml; n=10), was 10%. This was significantly decreased (p<0.05) compared to 52% survival observed for the second subgroup of patients with plasma TGFbeta(1) values close to HD (<3.00 ng/ml, n=19). CONCLUSION: We have performed a pilot study to determine the relationship between overall survival and TGF-beta(1) concentration in the blood of metastatic breast cancer patients. The survival was significantly reduced in the patients with elevated plasma TGF-beta(1) levels compared to that of the patients with plasma TGF-beta(1) levels close to normal. We propose that plasma TGF-beta(1) concentration may be a new tumour marker attributed to the presence of metastatic BC cells that may be used in selection of metastatic BC patients with poor prognosis.