High Farnesoid X Receptor (FXR) expression is a strong and independent prognosticator in invasive breast carcinoma.

Farnesoid X Receptor (FXR), a nuclear receptor superfamily member, is related with bile acids, glucose and lipids metabolism and recently with cancer. In the present study the clinical significance of FXR expression in invasive breast carcinoma was evaluated. FXR protein expression was assessed immunohistochemically on paraffin-embedded breast cancer tissues obtained from 115 breast cancer patients and was statistically analyzed with clinicopathological parameters, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression, as well as with tumor cells' proliferative capacity and overall and disease-free patients' survival. FXR positivity was noted in 91 (79.1%) and high FXR expression in 51 (44.3%) out of 115 invasive breast carcinoma cases. High FXR expression was significantly associated with smaller tumor size (p=0.0318) and increased tumor cells' proliferative rate (p=0.0375). Invasive breast carcinoma patients presenting high FXR expression showed significantly longer overall and disease-free survival times compared to those with low FXR expression (log-rank test, p=0.0052 and p=0.0058). In multivariate analysis, FXR expression was identified as independent prognostic factor of overall and disease-free patients' survival (Cox-regression analysis, p=0.0023 and p=0.0049, respectively). The present data support evidence that FXR may be implicated at the earlier stage of breast malignant disease progression, being a strong and independent prognosticator of favorable overall and disease-free survival in invasive breast carcinoma.

DNA repair mechanisms in colorectal carcinogenesis.

Colon cancer is among the most common cancers and the third cause of cancer deaths worldwide. If detected at an early stage, treatment might often lead to cure. The present review adduces the so far studied alterations in the expression of genes, as well as polymorphisms of genes engaged in DNA repair systems, with particular emphasis on indirect ones that are correlated with colorectal cancer. Such aberrations could be linked to an increased risk for the development of colorectal cancer and might serve as potential targets in the areas of prevention and therapy.

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Glucocorticoid receptor alpha and beta isoforms are not mutated in bipolar affective disorder.

The periodically hyperactive hypothalamic-pituitary-adrenal (HPA) axis in bipolar affective disorders, as well as the reported changes in the binding characteristics of the glucocorticoid receptor (GR), suggest the possible involvement of the GR in the aetiopathology of this disease. This was investigated by screening the coding sequences of both GR isoforms, GRalpha and GRbeta, for the presence of mutations. As a genetic predisposition has been implicated, we included in this study bipolar patients who were siblings. By RT-PCR of peripheral blood mononuclear cells from patients suffering from bipolar illness, using primers spanning the whole length of the GRalpha and GRbeta coding region and subsequent agarose gel electrophoresis, heteroduplex and sequence analyses, no GR mutations could be detected. Since glucocorticoid receptor activity can be modulated by agents other than the respective ligand (eg by growth factors, cytokines and stress signals), our results favor derangements in the modulation of GR activity by such agents and not in the primary structure of the receptor as aetiopathologic factors of bipolar disease.

Human papilloma virus DNA detection in oral lesions in the Greek population.

HPVs (Human Papilloma Viruses) are small DNA "epitheliotropic" viruses, implicated in cervical carcinogenesis, particularly the "high-oncogenic-risk" types HPV-16 and HPV-18. Data concerning oral carcinogenesis are however, contradictory. We examined the presence of HPV and subsequently HPV-16 and HPV-18 in 102 specimens of paraffin-embedded oral tissue blocks--81 oral squamous cell carcinomas and 21 oral hyperplasias--using PCR technique followed by agarose gel electrophoresis. Our results demonstrated that 49% (50/102) of the samples were HPV positive. Subsequent analysis of HPV positive lesions revealed 22% positivity for HPV-16 and 44% for HPV-18. HPV-18 was detected only in carcinomas, while HPV-16 was more abundant in papillomatous hyperplasias and in a small percentage of carcinomas. These findings may probably indicate a contributing role for HPV-18 as a potent co-carcinogen in oral epithelial carcinogenesis in the Greek population.

Cytogenetic analysis and RAS mutations in primary myelodysplastic syndromes.

Cytogenetic analysis was performed in 60 patients with primary myelodysplastic syndromes--diagnosed, treated, and followed in our department. In 41 cases, the presence of the NRAS mutation was also evaluated. The aim of this study was to evaluate the prognostic value of chromosomal abnormalities and NRAS mutation. The median age of the patients was 67 years (18-88 years), and the French-American-British classification was as follows: refractory anemia 26, refractory anemia with ring sideroblasts 4, refractory anemia with excess of blast cells 15, refractory anemia with excess of blast cells in transformation 3, and chronic myelomonocytic leukemia 12. Survival analysis was performed for the patients with a normal (n = 35), an abnormal (n = 25) karyotype and with a single (n = 15) or multiple (n = 10) cytogenetic abnormalities. Abnormal karyotypes were detected in 25 of the 60 patients (41.6%). Fifteen of these patients had a single and 10 had two or more lesions. The median survival of the patients with a normal (33.1 months) and with an abnormal (36.5 months) karyotype was not significantly different. Patients with multiple lesions had a reduced median survival compared with patients with single anomalies (19.2 versus 39.7 months, p = 0.5). Patients with an abnormal karyotype progressed to acute leukemia more frequently compared with patients without lesions (36 versus 28.6%, p = 0.5). NRAS mutation was detected in 2 of 10 CMMoL patients studied and in none of the 31 patients with other types of myelodysplastic syndrome. Marrow blasts more than 10% significantly affected survival.

Immunohistochemical detection of p53 protein in HPV positive oral lesions.

Oral cancer provides a unique model system for the study of the multistep nature of cancer. The influence of viruses and tumor suppressor gene inactivation are of major importance in this HPVs are small oncogenic viruses which are implicated in epithelial carcinogenesis, and p53 is a tumor suppressor gene with a central role in the prevention of genomic injury. p53 protein detection is usually a synonym for p53 mutation. This study was designed to determine the immunohistochemical detection of p53 protein in HPV positive oral squamous cell carcinomas and hyperplastic oral lesions. p53 was detected in 50% (5/10) of HPV positive hyperplastic oral lesions and in 59.41% (22/39) of oral squamous cell carcinomas. These results indicate that HPV and p53 protein alterations frequently coexist in the lesions of our study and suggest that p53 mutation may be an early genetic event in oral carcinogenesis. Moreover, this coexistance reveals that other environmental carcinogens have a more prominent role in oral carcinogenesis, one that overrides the action of HPV.

K-ras mutation in Greek patients with poorly and moderately differenciated tumours of the lower intestinal tract.

The K-ras protooncogene is activated via mutations in approximately 40% of primary colorectal adenocarcinoma. This finding suggests that these genetic alterations are important events in the genesis of colon cancer and should be correlated with other parameters in order to infer some conclusions relevant to the etiology and the pathogenesis of this type of cancer. In our study we examined whether the incidence of K-ras mutations detected in 23 samples with colorectal adenocarcinomas, was related to different anatomical sites within the lower intestinal tract (transverse colon, descending colon and rectosigmoid region) and also the correlation between K-ras mutations and depth of invasion, level of tumour cell differentiation and metastasis to the regional lymph nodes. For this study the critical regions for the activation of the K-ras protooncogene were amplified by the PCR technique and the particular sequences analysed, after their membrane transfer, by differential hybridization with selected synthetic oligonucleotides. Our results demonstrated that 39% of the adenocarcinomas examined contained point mutations, and 66.6% of these were located in the second position of K-ras codon 12, whereas the other 33.3% were located in the second position of codon 13. 77.8% of the mutations were located at the rectosigmoid region and the relevance of the mutations was higher in poorly differenciated tumours. The depth of invasion was associated with the presence of a mutation whereas no correlation was found between the presence of a mutation anb the regional lymph node metastasis.

Human papillomaviruses in cervical cancer: II. Application of PCR and in situ hybridization and HPV significance.

Human papillomaviruses especially type 16 and 18 are associated with cervical cancer. These viruses encode transforming proteins which are capable of binding and form complexes with p53 protein. Tumour-tissue samples from women with cervical cancer as well as normal controls were analysed for the presence of HPV and the expression of p53 by in situ hybridization and PCR analysis. The results showed that HPV 16 and 18 predominated in all pathological conditions and that p53 expression was related to the presence of certain types of HPV. The significance of HPV in cervical carcinoma is well demonstrated.

A rapid method for the screening and typing of high risk HPVs using molecular biology techniques.

To date over 60 different human papilloma virus (HPV) types have been described and novel HPV genomes are still being identified. The identification and taxonomy of papilloma viruses has become increasingly complex However, some types, especially HPV-16, -18 and to a lesser extent HPV-31 and -33, which are found in a high proportion of invasive cervical cancers and their metastases, are classified as high risk types For preventive reasons it is important to identify and classify the different HPV types in clinical specimens. Many of the methods used until recently are cumbersome. In this paper we use molecular biology techniques which permit a rapid screening and typing of high risk HPVs in clinical specimens. The screening procedure is based on the very sensitive method of polymerase chain reaction. Using a set of general primers derived from the E1 open reading frame, which anneal to a large variety of human papilloma virus DNA, we can classify samples into positive or negative for the presence of HPV sequences in a single step. The typing of the high risk HPV types is achieved by restriction enzyme analysis using the endonuclease Alu I which cleaves each high risk HPV type at different sites, thus permitting the easy identification of each type after agarose gel electrophoresis.

Comparative study of mutagenesis by O6-methylguanine in the human Ha-ras oncogene in E. coli and in vitro.

Single residues of O6-methylguanine (O6-meG) were introduced into the first or second position of codon 12 (GGC; positions 12G1 or 12G2, respectively) or the first position of codon 13 (GGT; position 13G1) of the human Ha-ras oncogene in phage M13-based vectors. After transformation of E.coli, higher mutant plaque frequencies (MPF) were observed at 12G1 and 13G1 than at 12G2 if O6-alkylguanine-DNA alkyltransferase (AGT) had been depleted, while similar MPF were observed at all three positions in the presence of active AGT. Taken together, these observations suggest reduced AGT repair at 12G2. Kinetic analysis of in vitro DNA replication in the same sequences using E. coli DNA polymerase I (Klenow fragment) indicated that variation in polymerase fidelity may contribute to the overall sequence specificity of mutagenesis. By constructing vectors which direct methyl-directed mismatch repair to the (+) or the (-) strand and comparing the MPF values in bacteria proficient or deficient in mismatch repair and/or AGT, it was concluded that, while mutS-mediated mismatch repair did not remove O6-meG from O6-meG:C pairs, this repair mechanism can affect O6-meG mutagenesis by repairing G:T pairs generated through AGT-induced demethylation of O6-meG:T replication intermediates.

Haplotype VI associated beta + thalassaemia intermedia. First Italian case.

We report the study of a family originating from eastern Sicily with mild beta thalassaemia intermedia which is similar both at a molecular level and in clinical form to that called "beta + thalassaemia intermedia-Portuguese type". Our patients were homozygous beta + thalassaemics with high HbA2 and low HbF levels. The mild clinical course was as a result of their age and because regular blood transfusion was established only in adulthood. All of the heterozygote parents were asymptomatic with a blood picture and haemoglobin pattern typical of beta thalassaemia. Studies at a molecular level revealed no abnormalities in the beta-like globin gene cluster and excluded the presence of a deletional form of alpha thalassaemia. Restriction enzyme site polymorphisms around the beta gene cluster showed that all patients were homozygous for the haplotype described as VI. Comparison of these homozygous haplotypes with the Portuguese ones revealed a clear difference in the polymorphic Pvu II site. In all Sicilian homozygous cases, this site was present on one chromosome and absent on the other. Therefore our hypothesis is that Portuguese beta + thalassaemia intermedia is different from the Sicilian type.

A leftward deletional alpha+ thalassemia found in East Sicily in conjunction with heterozygous beta-thalassemia.

Two types of alpha+ thalassemia (-alpha l) have been described, respectively termed leftward and rightward, which correspond to nonhomologous crossing-over in different homology zones X and Z within the alpha-globin gene cluster. Up to now the leftward type has been described only in Asiatic populations, whereas the rightward type is universally distributed. We report here a first case of leftward deletion observed in a Sicilian male. This raises the question of an identical or not crossing-over event.

Characterization of weak alleles at the DIA1 locus (Mustapha 1, Mustapha 2, and Mustapha 3) in the Algerian population.

One percent of the Algerian population carries a weak allele at the DIA1 locus, responsible for a 50% decrease of red cell soluble cytochrome b5 reductase activity. Quantitative abnormalities of the soluble and the membrane-bound enzyme have been investigated in the red cells and in the leukocytes of seven subjects considered to be heterozygous at the DIA1 locus. Conventional electrophoretic or isoelectrophoretic studies did not show any qualitative abnormality. However, continuous titration obtained by combined IEF-electrophoresis displayed in five out of seven subjects a discrete abnormal line on the titration curve compatible with an Arg leads to His substitution. In fact, at least three types of weak alleles could be defined by combining the qualitative and quantitative results obtained with the erythrocyte (soluble and membrane-bound) and leukocyte enzyme. We call these subgroups DIA Mustapha1, DIA Mustapha2, and DIA Mustapha3.