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Fine-needle aspiration cytology (FNC) is the primary means to distinguish benign form malignant nodules. Aim of this study is to evaluate the diagnostic value of BRAF(V600E) and RET/PTC oncogenes in a large cohort of thyroid nodules with inconclusive FNC. We searched for BRAF(V600E) and RET/PTC in 299 thyroid nodule aspirates then removed by surgery. RET/PTC demonstrated a poor specificity. The search for BRAF(V600E) demonstrated to be useful in 25 cases, identifying a PTC in 2 false negative, 2 inadequate, 11 indeterminate and 10 suspicious FNC. Detection of BRAF(V600E) revealed to be a useful tool to refine inconclusive cytology.

PCR protocols for immunoglobulin heavy chain (IgH) gene rearrangements amplification make easy the NHL-B identification. In this study we analyzed PCR products by Capillary Electrophoresis (CE) and GeneScan (GS) software, wich offers clear advantages over the conventional methods such as agarose gels (AGGE), characterized by hight rate of false negative and false positive results. We suggested some criteria--not included in previous NHL-B issues--useful to a correct analysis of results in GS. Since 2003, we collected 2,977 samples (2,770 peripheral blood and bone marrow, and 207 tissues) for GS analysis from NHL-B patients. At beginning PCR products were detected by both AGGE and CE. FR2 and FR3 VH regions were amplified by PCR seminested; together with Bcl-6 "housekeeping" gene from the same sample, as marker of DNA quality and PCR efficiency. Bcl-2/IgH and Bcl1/IgH traslocations were also analyzed for follicular and mantle cells lymphomas respectively. Resolution and sensitivity tests, developed with serial diluitions of clonal products in water and in DNA from healthy individuals, showed for GS 1% of resolution limit (3% AGGE) and 0.5% of sensitivity (5% AGGE). Our criteria for correct interpretations of results are: a) use of "house-keeping" gene Bcl-6; b) costant reference scales for hight and molecular weight; c) clonal peak at least twice higher than adiacent peaks; d) position of clonal peak (central or eccentric) as regards to policlonal peaks distributions. e) peaks features for oligoclonal or biallelic rearrangements evaluation. GS is an ideal method for detecting IgH rearrangements and some characteristic traslocations. The precise determination of the size of the PCR product can be used for the minimal residual disease evaluation. Moreover, it allows semi-quantitative resolution of fragments only one base different in size and may be more objective than gel-based methods.

We investigated the immunohistochemical espression of bcl-2 and the genetic assessment of the bcl-2 gene in relation to responsiveness to first line chemotherapy and to the clinical outcome in advanced ovarian carcinoma patients. We have compared 17 patients, with FIGO stage III C, ovarian serous carcinomas, G3, living with no evident disease five years after primary surgical treatment; to 19 patients who had died of progression of disease no later than two years after primary surgical treatment. The correlation of bcl-2 expression with the survival and the clinical responsiveness to chemotherapy, were analysed with the logistic regression. We observed a bcl-2 expression in tumor cells in 25% of the cases. Molecular genetic analysis of the bcl-2 gene was performed for all the bcl-2 immunohistochimical positive cases. No traslocation t(14;18)(q32;q21) of the gene bcl-2 were found. The bcl-2 over-expression was found to be a significant independent predictor of responsiveness to chemotherapy (p = 0.04), but it was not correlated with the overall survival of the ovarian cancer patients. The prognostic value of bcl-2 espression may help in the management of ovarian cancer patients permitting the selection of more aggressive first line chemotherapy. In addition, the knowledge of the molecular mechanism, which is responsible of the over-expression of bcl-2, may help in the understanding of mechanisms responsible for chemoresistance. Further studies in this area will help clarify this therapeutic possibility.

The authors discuss the importance that molecular medicine has assumed in recent years. Molecular methodologies have clearly demonstrated that immunological diversity is based fundamentally on the rearrangement of the genes encoding antigen B and T cell receptors. The importance of oncogenes, and their translocation in tumoral pathologies is emphasized, a case in point being the alterations observed in chronic myeloid leukemia and acute promyelocytic leukemia and their implication for innovative therapy. The importance of prothrombin and factor V genetic-molecular alterations in thromboembolic pathology and of the activation of calcineurin phosphatase or other intracellular signal regulator molecules during cardiac insufficiency genesis is also discussed. Particular attention is paid to progress regarding the socially important Alzheimer's syndrome, and the diagnosis of endocrine tumors. Moreover, the authors believe that the identification of new endocrine nuclear receptors, "orphans" of hormonal ligands, will open up interesting prospects--even therapeutic--in endocrinology. The authors conclude by reviewing the therapeutic prospects for immunodeficiency syndromes and malignant tumors, offered by new gene therapy methodologies. They also discuss recent results of studies on the aging process which, until not many years ago, appeared adventuristic. Today they are opening prospects of great interest.

Unlabelled: The application of PCR to detect monoclonality at the immunoglobulin heavy chain gene locus in FNA cytologic specimens was evaluated., Materials and Methods: Immunoglobulin heavy chain gene rearrangement analysis was performed by a nested PCR for the VDJ region on 27 lymph nodes FNA paraffin embedded by cell blocking technique specimens (1 suspicious of NOS lymphoma, 1 Hodgkin's disease, 6 B cell non Hodgkin's lymphomas, 10 reactive hyperplasias)., Results: A monoclonal band was seen in 13 of 17 suspicious or positive malignant cases. The other 4 specimens gave a policlonal pattern. Specimens from reactive lymph nodes produced policlonal bands in 8 cases, no product in 1 case and 1 specimen gave a monoclonal band. When needed, the results of PCR examinations were compared with the subsequent histologic evaluation of the same lymph-node., Conclusions: From our experience we can see that clonal analysis by PCR based IgH gene rearrangement analysis can be applied to FNA material and can be useful in diagnosis.

Unlabelled: The quantitation of DNA and growth fraction in the different step from dysplastic to neoplastic process in large bowel is the aim of this study. 70 colonic polyps were studied. The fresh specimens were processed and DNA analysis was carried out using a Partec CA II flow cytometer and the growth fraction was tested with KI-67 monoclonal antibody. The percentage of S-phase cells has been calculated with the Multicycle program. Our results demonstrated that 7 adenomas were tubulo-villous with mild dysplasia, 39 with mild-moderate dysplasia, 1 with severe dysplasia, 5 were polypoid carcinomas, 2 juvenile polyps, 1 polypoid leiomyoma, 1 inflammatory fibroid polyps. DNA analysis showed a diploid DNA content in non adenomatous polyps, in all adenomas with mild dysplasia, in 37 with mild-moderate dysplasia, in 8 cases with moderate-severe dysplasia and 1 cancer. Aneuploidy was discovered in 2 cases with mild-moderate dysplasia, in 6 cases with moderate-severe dysplasia, in the case of severe dysplasia and in 4 cases of carcinomas. Best indexes of linear correlation (Pearson's r) has been found between S-phase and DNA index (r = .75) and between S-phase and KI-67 (r = .82)., In Conclusion: 1) No relationship was found between DNA content and age, sex, size and location of polyps. 2) Aneuploidy is strictly related to moderate-severe grade of dysplasia therefore it is an important element in the development of adenomacarcinoma sequence. 3) DNA-index, S-phase and KI-67 are strictly related.

Squamous cell laryngeal carcinoma accounts for 1% of all cancer deaths and 95% of all laryngeal malignancies. It is most frequently found in smokers over 40 years of age. This neoplasm is presently the object of cytogenetic studies in an attempt to identify a specific chromosome pattern. In a study of 29 cases of malignant primary laryngeal tumor, Nawroz (1993) found a loss of alleles in different loci mapped in the short arm of chromosome 9 (9p) in more than two-thirds of the cases. In the same chromosome region, the loss of heterozygotes (LOH) was previously described in other neoplasms (leukemia, hematic tumors, melanomas). In an attempt to verify the predominant chromosome pattern and the loss of heterozygotes in chromosome 9, a cytogenetic, genetic-molecular study was performed on ten cases of laryngeal carcinoma. Among these subjects, two showed a hyperdiploid chromosome pattern (metaphase with more than 46 chromosomes per cell), five had a hypodiploid pattern (with less than 46 chromosomes per cell) while, for the remaining three cases, it was not possible to identify any metaphase. Numerous structural and numerical karyotype defects were found in chromosomes 1, 3, 4, 5, 9, 10, 13, 14, 16, 18, and Y. In 6 of the cases abnormality was found in chromosome 9 while in 10 it was apparently a homozygote. The study was performed with the use of fluorescent in situ hybridization (FISH) using a chromosome library specific for chromosome 9. A loss of the 9p segment could be found as a result of different types of alterations: deletion (case 1, 2, 5, 7); non uniform transfer between chromosome 2 and chromosome 9 (case 2); other transfers involving the 9p segment (case 1, 4, 5, 7, 10). In six cases, analysis was further detailed at the molecular level by means of DNA amplification methods (PCR) and electrophoresis on denatured 10% polyacrylammide gels. LOH was studied using a polymorphic system specific for the short arm of chromosome 9. Four of the cases examined showed LOH for the system used while one case (case 4) gave no information. Case 9 did not show any loss of alleles. The present study suggests that the loss of a DNA sequence on chromosome 9p is primary to the neoplastic progression in laryngeal cancer.

We have conducted a review of the literature on chemical exposures (known carcinogens), mutations in oncogenes/tumor suppressor genes and the occurrence of malignancies. The underlying hypothesis of the studies on this issue is that specific carcinogenic chemicals induce specific mutations in relevant genes ("fingerprints") and that the latter are involved in the causal pathway that leads to cancer. The confirmation of such hypothesis is conditioned by limitations of the available techniques, and by the design of the epidemiological studies. We discuss methodological problems inherent in the identification of oncogene mutations within studies in humans, such as the limited sensitivity of molecular techniques, which may lead to a bias toward the null. Although several studies--concerning mainly lung or bladder cancer and tobacco smoking or some occupational exposures--give support to the theory of "fingerprints", further evidence is warranted.

One of the problems connected to the anatomo-clinical evaluation of ependymomas consist in the difficulty of precisely grading these neoplasms and of making a prognosis. Traditional histopathologic procedures have proven themselves unsuitable to solve these biological problems of neoplastic growth: their solution requires the identification and responsiveness of molecules that are part of cellular kinetic processes such as antigen Ki67 and the granules of AgNOR as indexes of the rDNA transcriptional activity. With the aim of contributing to the knowledge of ependymomas' grading, the Authors performed qualitative and quantitative assays on 35 cases of benign ependymomas (by means of an image analyzer) using the nuclear antigen Ki67 and the AgNOR nucleolar areas. The results thus obtained allow to draw the following conclusions: 1) The biological validity of the labeling of antigen Ki67 and the quantification of argentophilic granules in the NOR areas has once more been confirmed. 2) The expressiveness and biological significance of antigen Ki67 is finer than what can be derived from the count of AgNOR granules. 3) The level of diagnostic reliability for the differentiation between and the corresponding malignant forms is optimal; such a level falls considerably when one wishes to grade levels of benignity of malignity. 4) Such drawbacks are likely to be due to the action of heterogeneous factors which are often unrelated to the neoplasm, such as poor oxygenation, blood stream alterations, necrosis, phlogosis, nutritional conditions of the cell populations etc.

The oncogenic capacities of RAS family genes (Ha-ras, Ki-ras, and N-ras) are usually activated by point mutations in the conserved regions (codons 12, 13, and 61), resulting in single amino acid substitution in the specific proteins (p21). In order to verify the involvement of RAS genes in dog mammary tumors we analyzed the genomic DNA from 20 mammary tumors of dog by means of the Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) method and the direct genomic sequencing. The absence of point mutations in the "hot spots" of RAS genes suggests a lack or a low frequency of such a pattern of RAS genes activation in dog mammary tumors. The results are also in agreement to what reported in human mammary tumors. However, the presence of genetic alterations in other functional areas of the RAS genes or other mechanisms of activations cannot be ruled out.

Of 567 children with neuroblastoma diagnosed between November 1984 and May 1993 in 21 Italian institutions, 235 (41%) have been evaluated for MYCN oncogene amplification. The amplification (3 or more copies of the gene) was found in 39 patients (17%) and was more frequent in patients aged more than one year, abdominal primary site of the tumor, advanced stages, normal urinary excretion of vanillylmandelic acid (VMA), and high level of LDH, NSE and ferritin. The five-year survival of the 235 patients (62%) was significantly better in patients with normal copy number of MYCN (69% versus 29%). By correlating genomic amplification with clinical and biochemical characteristics, MYCN amplification was found associated with a worse prognosis even when patients were subdivided for age (under and above one year), disease extension (localized operable, localized but inoperable, and disseminated) with exception for Stage IV-S, VMA and homovanillic acid excretion, serum levels of NSE and ferritin, but not of LDH. These data confirm the unfavourable prognostic meaning of MYCN amplification, but are unable to define if it represents a new independent variable.

The Authors analyse their experience about 387 cases operated for rectal cancer. The choice of the operation was not related to tumour's features, but was related to the location of the neoplasia. The safety margin of rectal resection was reduced up to a minimum of 2 cm. in lower tumours, where it was not possible to meet this condition an abdomino-perineal resection was performed. A lumbo-aortic lymphadenectomy with high ligature of inferior mesenteric artery was always performed; in 17 cases it was extended to the pelvic nodes. In all the patients the whole mesorectum was removed and a wide pelvic dissection was performed to avoid the so called cone effect. Since 1991 all the B2-C patients undergo preoperative radiotherapy. Recurrence rate and survival are similar both after abdomino-perineal and sphincter-saving resections. Among the different risk factors, the Authors have found a statistically significant relation only with the stage. The Authors, therefore, conclude that sphincter-saving resection is the best surgical procedure because of the god quality of life. They stress the importance of a lumboarotic lymphadenectomy with high ligature of inferior mesenteric artery. As far as the complementary therapy is concerned, the discussion is still open, especially about the timing and the choice of the adjuvant treatment.

We have analysed the nuclear DNA content in a series of 33 thyroid tumours (18 carcinomas and 15 adenomas) and, for comparison, in 189 nodular strumas and 17 tissue samples of normal thyroids, calculating cell cycle parameters and presence of aneuploid DNA. Cell suspensions were prepared for each of three tissue fragments taken for different areas of the surgical specimens. Cell subpopulations with aneuploid DNA content were present in 39% of carcinomas, 27% of adenomas, 20% of strumas, but were absent in the samples from normal thyroids. The presence of hyperdiploid DNA was much more frequent than that of hypodiploid DNA in all pathologic tissues. The mean proliferation index was 14.1 in the carcinomas, 6.6 in the adenomas, 7.1 in the strumas and 6.1 in normal thyroid tissues. The percent of histograms with a coefficient of variation of the G0-G1 peak greater than 5 was highest in the carcinomas (26%). The significance and implications of the reported data are discussed for the interpretation of thyroid neoplastic pathologies.