The 811 C/T polymorphism in the 3' untranslated region of the selenoprotein 15-kDa (Sep15) gene and breast cancer in Caucasian women.

The 15-kDa selenoprotein (Sep15) is a selenocysteine-containing oxidoreductase in the endoplasmic reticulum that participates in disulfide-bond formation and protein folding control. The 3'-untranslated region (3'-UTR) contains two exclusively linked, polymorphic sites at positions 811 (C/T) and 1125 (G/A), which result in two functional haplotypes: 811C/1125G or 811T/1125A. The 811T/1125A variant occurs significantly more often in African-Americans as compared to Caucasians and has been linked to increased breast cancer risk in black women. We studied the 811C/T (rs5845) Sep15 gene polymorphism in 182 Caucasian women-83 breast cancer cases and 99 healthy controls-by pyrosequencing and polymerase chain reaction. Associations between allelic variants and clinico-pathological variables (e.g., age, stage of disease, tumor type, grading, and receptor status) were investigated. The genotype distribution in breast cancer patients (CC 63.9 %, CT 33.7 %, TT 2.4 %) and controls (69.7 %, CT 28.3 %, TT 2 %) showed no significant difference (OR 0.77, 95 % CI 0.41-1.42, p = 0.4). The overall low prevalence of the T allele was in accordance with that reported for Caucasians in previous studies. There was no significant association between 811C/T Sep15 polymorphism and any of clinico-pathological parameters. In conclusion, we are the first to report on 811C/T SEP 15 polymorphism in white breast cancer patients. Genotype variation within the 3'-UTR of the SEP 15 gene showed no association with breast cancer risk or clinico-pathological parameters in Caucasian women.

Biochip-based detection of KRAS mutation in non-small cell lung cancer.

This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC) tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip). Biochip hybridization identified 17 (21%) samples to carry a KRAS mutation of which 16 (33%) were adenocarcinomas and 1 (3%) was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples.

Clone-specific expression, transcriptional regulation, and action of interleukin-6 in human colon carcinoma cells.

BACKGROUND: Many cancer cells produce interleukin-6 (IL-6), a cytokine that plays a role in growth stimulation, metastasis, and angiogenesis of secondary tumours in a variety of malignancies, including colorectal cancer. Effectiveness of IL-6 in this respect may depend on the quantity of basal and inducible IL-6 expressed as the tumour progresses through stages of malignancy. We therefore have evaluated the effect of IL-6 modulators, i.e. IL-1beta, prostaglandin E2, 17beta-estradiol, and 1,25-dihydroxyvitamin D3, on expression and synthesis of the cytokine at different stages of tumour progression. METHODS: We utilized cultures of the human colon carcinoma cell clones Caco-2/AQ, COGA-1A and COGA-13, all of which expressed differentiation and proliferation markers typical of distinct stages of tumour progression. IL-6 mRNA and protein levels were assayed by RT-PCR and ELISA, respectively. DNA sequencing was utilized to detect polymorphisms in the IL-6 gene promoter. RESULTS: IL-6 mRNA and protein concentrations were low in well and moderately differentiated Caco-2/AQ and COGA-1A cells, but were high in poorly differentiated COGA-13 cells. Addition of IL-1beta (5 ng/ml) to a COGA-13 culture raised IL-6 production approximately thousandfold via a prostaglandin-independent mechanism. Addition of 17beta-estradiol (10-7 M) reduced basal IL-6 production by one-third, but IL-1beta-inducible IL-6 was unaffected. Search for polymorphisms in the IL-6 promoter revealed the presence of a single haplotype, i.e., -597A/-572G/-174C, in COGA-13 cells, which is associated with a high degree of transcriptional activity of the IL-6 gene. IL-6 blocked differentiation only in Caco-2/AQ cells and stimulated mitosis through up-regulation of c-myc proto-oncogene expression. These effects were inhibited by 10-8 M 1,25-dihydroxyvitamin D3. CONCLUSION: In human colon carcinoma cells derived from well and moderately differentiated tumours, IL-6 expression is low and only marginally affected, if at all, by PGE2, 1,25-dihydroxyvitamin D3, and 17beta-estradiol. However, IL-6 is highly abundant in undifferentiated tumour cells and is effectively stimulated by IL-1beta. In case of overexpression of an IL-6 gene variant with extreme sensitivity to IL-1beta, massive release of the cytokine from undifferentiated tumour cells may accelerate progression towards malignancy by paracrine action on more differentiated tumour cells with a still functioning proliferative IL-6 signalling pathway.

Epidermal growth factor receptor (EGFR) mutation does not correlate with platinum resistance in ovarian carcinoma. Results of a prospective pilot study.

BACKGROUND: Different studies have demonstrated epidermal growth factor receptor (EGFR) status as an independent prognostic factor for ovarian cancer (OC). Recent studies in non-small cell lung cancer suggest that the presence of a clinical response to tyrosine kinase inhibitors correlates with somatic mutations in the kinase domain of EGFR, exons 18-21. For patients with OC, data are not available on EGFR gene mutation. MATERIALS AND METHODS: Shock-frozen samples from 32 patients with OC were screened for L858R deletion mutations of EGFR within exon 21 of the kinase domain and 15 bp deletion in exon 19. Additionally, nine commercially available OC cell lines and 32 established OC lines were analysed. RESULTS: In cell lines, as well as in tumor samples, stratified to platinum-free therapy interval, no mutation of the EGFR gene was observed. CONCLUSION: Mutations in the kinase domain of the EGFR, exons 19 and 21, are absent or very infrequent in patients with OC.

The neuronal guidance cue Slit2 induces targeted migration and may play a role in brain metastasis of breast cancer cells.

Cell migration is essential in many diverse processes ranging from embryonic development to wound healing and immune response. Cancer cells have recently been shown to utilize chemoattraction mechanisms mediated by chemokines and their respective receptors, e.g., the CXCL12/CXCR4 pathway normally found in leukocytes. Here we show that Slit2, a secreted protein signaling through the Roundabout (Robo) receptor as a chemorepellent in axon guidance and neuronal migration, acts as a potent chemoattractant for breast cancer cells. Comparing cell lines specifically metastasizing to either brain or bone, we found significant differences in their responses to CXCL12 and Slit2 treatments, suggesting a role for Slit/Robo signaling in brain metastasis.

Five genes from chromosomal band 8p22 are significantly down-regulated in ovarian carcinoma: N33 and EFA6R have a potential impact on overall survival.

BACKGROUND: Loss of heterozygosity on chromosomal band 8p22 is a common event in several epithelial tumors including ovarian carcinoma. So far, no clear evidence for a tumor suppressor gene (TSG) in this region has been found. METHODS: On the basis of publicly available expression data in ovarian tissues, the authors selected the eight most noteworthy genes from 8p22 (DLC1, N33, ZDHHC2, FLJ32642, PDGFRL, MTSG1, PCM1, and EFA6R) for a detailed expression analysis in 58 primary ovarian carcinoma tissues and in 38 ovarian cancer cell lines by using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Expression data were correlated to various clinicopathologic characteristics and survival. RESULTS: Two genes showed a significantly (P< 0.05) lower expression in grade 3 tumors compared with tumors of lower grade (N33) or compared with normal controls and tumors with lower grade (EFA6R). Expression of N33 and EFA6R seems to have an impact on survival, in particular when the combined expression of both genes was used as predictive factor (P< 0.003). In addition, N33 and EFA6R showed a complete loss of expression in several ovarian cancer cell lines. Three genes (FLJ32642, MTSG1, and PCM1) had a significantly (P< 0.001, P< 0.004, and P< 0.001) lower expression in primary ovarian carcinoma compared with controls (ovarian tissues and cysts). CONCLUSIONS: Two to five new potential tumor suppressor or antagonizing gene candidates (N33 and EFA6R with impact on survival, and potentially FLJ32642, MTSG1, and PCM1) for ovarian carcinoma, were identified from the chromosomal band 8p22 and are promising candidates for further functional analysis in ovarian carcinoma.

HMGA2 is associated with invasiveness but not a suitable marker for the detection of circulating tumor cells in breast cancer.

Previously, the human high mobility group protein member HMGA2 mRNA was reported to be expressed in peripheral blood of patients with breast cancer, but not in healthy individuals. Expression of HMGA2 in blood was suggested to be an independent indicator of poor prognosis in metastatic breast cancer. These very promising findings propose HMGA2 as a potential marker for the detection of circulating tumor cells in peripheral blood. Therefore, we analyzed peripheral blood specimens from healthy controls and patients with breast tumors for HMGA2 expression using TaqMan real-time RT-PCR to test if HMGA2 is a suitable marker for the early detection of breast cancer and monitoring therapy response in peripheral blood. Furthermore, we examined the possible involvement of HMGA2 expression in invasion investigated by an in vitro invasion assay using established breast cell lines. HMGA2 expression was detected in peripheral blood of breast cancer patients as well as of healthy individuals. No significant association of HMGA2 expression with any clinical or histopathological data was apparent. However, there was a significant correlation of HMGA2 expression in invasive and non invasive breast cell lines (p=0.0056). Although, HMGA2 obviously contributes to invasion it is not a specific marker for the detection of circulating tumor cells in peripheral blood.

Evaluation of MUC1 splice variants as prognostic markers in patients with ductal carcinoma in situ of the breast.

Despite intense research in the field of breast cancer it still remains the most common cancer in women in the Western world. A decreasing trend in mortality was mainly achieved by improved early detection which led to an increased incidence of ductal carcinoma in situ (DCIS) of the breast. For the patient's prognosis and the administration of a patient-tailored therapy strategy it is crucial to identify diagnostic and prognostic markers for high-risk DCIS patients. MUC1 is associated with tumour aggressiveness in human breast cancer. Recent studies used MUC1 splice variant A to identify malignant thyroid cancer. In the present study we have examined the usefulness of MUC1 splice variants as prognostic markers in DCIS. We used laser capture microdissection of paraffin-embedded tissue to isolate RNA from isolated tumour cells and determined the MUC1 splice variant distribution by RT-PCR. In the majority of cases variant B was more highly expressed than variant A. This was true for pure DCIS (66%) as well as for DCIS with adjacent invasive cancer (66%). In 7 out of 18 cases (38%) of pure DCIS variant A was not expressed at all. In DCIS with adjacent invasive cancer only 2 samples out of 12 showed this expression pattern (16%). The situation that variant A was more highly expressed than B, or that variant B was not expressed at all, was similar for pure DCIS (27%) and for DCIS with adjacent invasive cancer (33%). The present study describes the differences of MUC1 splice variant expression in pure DCIS compared to DCIS with adjacent invasive cancer. A discriminating pattern of MUC1 splice variants could not be demonstrated.

Analysis of an interleukin-6 gene promoter polymorphism in women with endometriosis by pyrosequencing.

Interleukin (IL)-6 has been implicated in the etiology of endometriosis. A single nucleotide polymorphism (SNP) at position -174 in the IL-6 gene promoter appears to influence IL-6 transcription rates in vitro and basal IL-6 levels in vivo. We determined the genotype and the allele frequencies of the -174 IL-6 promoter polymorphism and the corresponding IL-6 serum levels in women with endometriosis. The pyrosequencing technique was used to assess the IL-6 genotypes in 94 women with histologically confirmed endometriosis (study group). A series of 70 healthy women without history of uterine disease served as clinical controls (control group).Allele frequencies for the G allele among women with and without endometriosis were 59.6% and 55.0%, respectively (P =.430; odds ratio [OR] 0.83, 95% confidence interval [CI] 0.53, 1.29). Homozygotes for the protective allele C were present in 17.0% of women with endometriosis and in 18.6% of controls were homozygous for the protective allele C (P =.797; OR 0.90, 95% CI 0.40, 2.02). When patients with various disease manifestations were compared, we found an association between the -174 G allele and chocolate cysts (P =.037). Serum levels of IL-6 were significantly higher in women with endometriosis than in controls (P <.001), with highest levels in women with chocolate cysts. There was no association between serum IL-6 levels and IL-6 genotype. The IL-6 promoter polymorphism -174 G/C does not contribute significantly to overall disease susceptibility but does predispose the carrier to distinct endometriosis with chocolate cysts. A genetically determined high IL-6 response might play a pathogenic role in this disease condition.

Tumor necrosis factor-alpha promotor polymorphisms and endometriosis.

To explore whether having the mutant tumor necrosis factor (TNF)2 (G-308*A) and TNFA-A (G-238*A) alleles in the TNF-alpha gene promotor region is higher in women with endometriosis, we determined the respective genotype and allele frequencies in a retrospective case-control study. Polymerase chain reaction was performed to identify the G-308A and G-238A promotor polymorphisms in 92 women with surgically and histologically confirmed endometriosis. A series of 69 healthy women without a history of endometriosis served as clinical controls. The allele frequencies of the TNF2 polymorphism were 0.13 and 0.16 in women with endometriosis and in the control group, respectively, and the frequencies of the TNFA-A polymorphisms in women with endometriosis and in the control group were 0.04 and 0.05, respectively, with no significant difference between the study and control groups. The TNF2 polymorphism was present in the homozygous form (TNF(2/2)) in 4.3% of women with endometriosis and in 2.9% of controls (P=.7). No TNFA-A homozygotes (TNFA(A/A)) were detected. We studied TNF-alpha promotor gene variants among women with endometriosis and found that having the G-308A TNF-alpha and the G-238A TNF-alpha polymorphism was not associated with endometriosis in a white population.

Human progesterone receptor gene polymorphism PROGINS and risk for breast cancer in Austrian women.

A germline TaqI restriction fragment length polymorphism in the intron G of the progesterone receptor (PR) gene designated PROGINS was described to be associated with an increased risk for ovarian carcinoma. It was supposed that the PR isoform A protein associated with PROGINS has an increased stability and therefore, a higher transcriptional activity. This may cause an inadequate control of estrogen receptor (ER) and PR B isoform and lead to the increased risk of tumor development. On the other hand, loss of heterozygosity (LOH) of the chromosomal region 11q22-23, where the PR gene is located, was frequently observed in breast cancer, suggesting the presence of a tumor suppressor gene in this region. Recently, it was reported that PROGINS is associated with a decreased risk for breast cancer. In order to examine if PROGINS is associated with an alteration of the risk for breast cancer, we examined PROGINS in 155 sporadic breast cancer patients in Austrian women and in a control group of 106 healthy volunteers. LOH affecting the PR gene was also analyzed in the tumor patients. No statistically significant difference was found for the frequencies of the PROGINS carriers (23.2%) in the Austrian breast cancer patients and in the healthy control group (26.4%), indicating that PROGINS is not associated with either an increased or a decreased risk for breast cancer. Furthermore, no associations between the PROGINS status and the protein levels of ER and PR, clinical data like tumor type, differentiation grade, tumor size, and the nodal status as well as the age of the patients were found. There was also no significant difference in the frequency of LOH affecting the PR gene in the PROGINS carriers and non carriers, demonstrating that LOH at PR gene is not associated with the PROGINS status.

Genetic alterations in endometrial hyperplasia and cancer.

Putative precursors of endometrial cancer such as complex endometrial hyperplasia with atypia have been described to be monoclonal and considered to be genetically related. In order to identify a genetic marker that could serve as a putative predictor of endometrial cancer we analyzed 14 endometrial hyperplasia and 29 endometrial cancer samples for instabilities and loss of heterozygosity (LOH) in microsatellite sequences. Deletions on the short arm of chromosome 8 were frequently detected in both endometrial hyperplasia and cancer samples, suggesting that these deletions are early events in the development of endometrial cancer.