Preoperative Assessment of TERT Promoter Mutation on Thyroid Core Needle Biopsies Supports Diagnosis of Malignancy and Addresses Surgical Strategy.

In the last decade, several molecular markers have been proposed to improve the diagnosis of thyroid nodules. Among these, mutations in the telomerase reverse transcriptase (TERT) promoter have been correlated to malignant tumors, characterized by highest recurrence and decreased patients' survival. This suggests an important role of TERT mutational analysis in the clinical diagnosis and management of thyroid cancer patients. The aim of the study was to demonstrate the adequacy of core needle biopsy (CNB) for the preoperative assessment of TERT mutational status, to reach a more accurate definition of malignancy and a more appropriate surgical planning. Indeed, CNB is gaining momentum for improving diagnosis of thyroid nodules deemed inconclusive by fine needle aspirate (FNA). The study included 50 patients submitted to CNB due to inconclusive FNA report. TERT mutational status was correlated with BRAF mutation, definitive histology, and post-operative TNM staging of the neoplasia. C228T mutation of the TERT promoter was reported in 10% of the papillary carcinomas (PTC) series. When compared with final histology, all cases harboring TERT mutation resulted as locally invasive PTCs. The prevalence of TERT mutated cases was 17.6% among locally advanced PTCs. TERT analysis on CNB allows the assessment of the pathological population on paraffin sections before DNA isolation, minimizing the risk of false negatives due to poor sampling that affects FNA, and gathering aggregate information about morphology and TERT mutational status. Data indicating a worse outcome of the tumor might be used to individualize treatment decision, surgical option, and follow-up design.

Phenotypic changes of the thyrocyte membrane in papillary thyroid carcinoma. A three-dimensional study.

Aim of the study was to assess the presence of structural changes in the complex carbohydrate chains of thyroid epithelia undergoing neoplastic transformation. We investigated thyroid cells from neoplastic lesions using a panel of lectins with specific affinity for distinct carbohydrate residues. Sixty samples of thyroid tissue, including normal, hyperplastic and neoplastic lesions were obtained from surgical specimens and blindly evaluated with lectin stains. Confocal microscopy was used to obtain three-dimensional (3-D) images of the samples with a positive reaction. Wheat germ agglutinin (WGA) was consistently positive on the apical membrane of papillary thyroid carcinomas (PTC), was weakly expressed in follicular carcinomas (FC) and resulted negative in normal thyrocytes and in benign conditions. The 3-D microscopy model showed that the WGA staining pattern in light microscopy corresponds to a continuous layer on the luminal surface of both papillary and tubular structures of PTC cells. The other lectins under evaluation did not provide any significant result. In conclusion, in PTC the apical border of thyrocytes showed a strong, specific and consistent staining with WGA. These findings may be related to a modified interaction of thyroglobulin molecule with thyroid cell membrane and with the expression of molecules that are involved in the process of tumorigenesis and tumor progression.

Comparative evaluation of genome-wide gene expression profiles in ruptured and unruptured human intracranial aneurysms.

Few studies have evaluated the over or the underexpression of genes directly in samples of aneurysmal wall and extracranial pericranial vascular tissue to investigate the genetic influence in formation and rupture of intracranial aneurysms. We present the results obtained using the DNA microarray technique analysis on sample tissues collected during surgery. We collected and analyzed 12 aneurismal and 9 peripheral arteries (superficial temporal (STA) and middle meningeal artery (MMA) specimens from ruptured aneurysm group patients (13 cases), 10 aneurismal and 12 STA and MMA samples from unruptured aneurysm group patients (14 cases) and 5 STA and MMA artery specimens from control group patients (4 cases). Total RNA was isolated from samples and subjected to cDNA microarray analysis with the use of the human genome U133A GeneChip oligonucleotide microarray (Affymetrix, Santa Clara, CA), which allows to analyze a total number of 14,500 genes in the same time. For genes of interest, real-time RT-PCR was performed to confirm their expression level. Total RNA was isolated from samples and subjected to DNA microarray analysis with the use of the human genome U133A GeneChip oligonucleotide microarray, which allows to analyze a total number of 14,500 genes at the same time. For genes of interest, real-time RT-PCR was performed to confirm their expression level. Regarding ruptured aneurysms, genes were identified showing differential expressions (overexpressed or downregulated) pertaining to specific pathways, particularly those for the structural proteins of the extracellular matrix, members of matrix metalloproteinase (MMP) family (which resulted as being overexpressed) and genes involved in apoptotic phenomena. Particularly, real-time RT-PCR analysis confirmed the upregulation of MMP-2, MMP-9 and pro-apoptotic genes, such as Fas, Bax and Bid, and the downregulation of anti-apoptotic genes, such as Bcl-X(L) and Bcl-2. In a compared analyses of ruptured vs unruptured aneurysms, a different expression was also detected regarding gene coding the tissue inhibitor of matrix metalloproteinases 3 (TIMP-3), which appeared markedly downregulated in unruptured aneurysms, where its expression in unruptured aneurysms was similar to that observed in controls. Another gene differently expressed is nitric oxide synthase (iNOS), which appeared overexpressed in ruptured aneurysms when compared to unruptured aneurysms. Our study is the first, to our knowledge, that compares gene expression profiles (genoma-wide) in intracranial aneurysms. The results of our study suggest that the inhibitor of the metalloproteinase, the pathway of nitric oxide and the apoptotic process play a key-role in reducing the resistance of the arterial wall, that can result in formation and rupture of the intracranial aneurysms.

Borderline HER-2 breast cancer cases: histochemical versus real-time PCR analysis and impact of different cut-off values.

Seventy-one cases that had resulted borderline for HER-2 protein expression at conventional immunohistochemical assay (2+) were assessed for HER-2 gene amplification by real-time PCR and by FISH in accordance with the manufacturer's recommendations (gene amplification with ratio >or=2 in both methods). Thirty-three out of 71 cases (47%) resulted amplified at real-time PCR analysis, whereas 15 cases resulted positive at FISH (21%). Apparently, PCR was more sensitive than FISH in HER-2 determination, only 10 cases resulting amplified in both tests. When the mean ratio value obtained in all PCR experiments was adopted as threshold in determining HER-2 gene amplification, the apparent sensitivity of PCR was reduced but correlation between PCR and FISH results was dramatically increased. Furthermore, when the mean PCR ratio value observed in the FISH-positive group was chosen as threshold, the best agreement between PCR and FISH results was achieved. Therefore, we found that the proposed threshold ratio value of >or=2 is not accurate in separating HER-2 amplified and non-amplified cases. We suggest that the threshold ratio value in PCR tests should be determined in each laboratory using FISH controlled cases. Finally, above certain in-lab generated threshold values, PCR might be proposed as a highly predictive positive test in HER-2 assessment.

Apoptosis-related gene expression in benign prostatic hyperplasia and prostate carcinoma.

BACKGROUND: The aim of this study was to examine the expressions of the bcl-2, bax, fas and c-myc apoptosis-related genes in benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) to determine whether significant differences exist within each disease and between the two groups of patients. The correlation between gene expression and tumour diameter, stage, Gleason score and serum PSA was also investigated. PATIENTS AND METHODS: Tissue specimens from 51 cases of BPH and 27 cases of CaP were examined for bcl-2, bax, fas and c-myc expression by reverse transcriptase-PCR (RT-PCR). RESULTS: In BPH, bcl-2 and bax gave the weakest signals (p < 0.001). In CaP, bcl-2 was the least expressed gene (p < 0.001). In both patient groups, fas and c-myc were the most highly expressed genes (p < 0.05). Both bcl-2 and bax were expressed at higher levels in CaP than in BPH (p < 0.02). The bcl-2/bax ratio was lower in CaP than in BPH (p < 0.001). Bcl-2 was more highly expressed in high Gleason grade (> 7) tumours (p < 0.05). In the BPH group, bax showed a positive relationship with fas (p < 0.01), while the bcl-2 level inversely correlated with that of c-myc (p < 0.05). CONCLUSION: Our data showed that all the apoptosis-related genes were expressed in both BPH and CaP. The stronger expression of bax and the lower bcl-2/bax ratio observed in CaP may suggest a pro-apoptotic stimulus, while the higher bcl-2 levels appear to counterbalance the tendency to cell death.

Validity of thyroglobulin mRNA assay in peripheral blood of postoperative thyroid carcinoma patients in predicting tumor recurrences varies according to the histologic type: results of a prospective study.

BACKGROUND: The objective of the current study was to evaluate the ability of serum thyroglobulin mRNA assay in detecting local and distant recurrences in patients who underwent surgery for thyroid carcinoma. METHODS: Sixty-six consecutive patients were studied. One year after surgery, all patients underwent clinical examination and radioiodine scan, and a blood sample was taken for serum thyroglobulin (Tg) immunoassay and for Tg mRNA assay by reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from cells pellet and analyzed by RT-PCR using specific primers for Tg. RESULTS: Thyroglobulin mRNA was detected in 14 (21.2%) patients. Seven of 16 patients with elevated serum thyroglobulin had detectable Tg mRNA. Six of 30 (20%) patients with absent or minimal thyroid bed radioiodine uptake and 7 of 36 (19.4%) patients with significant thyroid bed uptake had detectable Tg mRNA. Among 5 patients with metastases, only 1 (20%) showed circulating Tg mRNA. Overall, the sensitivity, specificity, and accuracy of Tg mRNA assay in predicting the results of the (131)I whole-body scans was 25%, 80%, 25%, respectively. Fourteen of 53 (26.4%) patients with papillary thyroid carcinoma had detectable thyroglobulin mRNA whereas none of the patients with other histologic types did. The sensitivity, specificity, and accuracy of Tg mRNA assay in predicting the results of the (131)I whole-body scans in patients with papillary thyroid carcinoma was 100%, 75%, and 100%, respectively. Of note, the percentage of cases with detectable Tg mRNA was similar among patients who did not receive postoperative (131)I and those who had postoperative radioiodine treatment. CONCLUSIONS: The current study suggests that the validity of the Tg mRNA assay varies according to the histologic type of thyroid carcinoma and that this assay may play a role in the identification of metastatic disease in the subgroup of patients affected by papillary thyroid carcinoma but does not appear to be sensitive or active enough to direct clinical management.

Thymic disorders and myasthenia gravis: genetic aspects.

Myasthenia gravis (MG) is an autoimmune disease characterized by production of antibodies to acetylcholine receptor at the motor end-plate responsible for impairment of neuromuscular transmission. There is general agreement about the involvement of the thymus in the pathogenesis of MG, and thymic pathological changes are commonly found in MG patients. Genetic factors seem to play an important role in susceptibility to MG. As with other autoimmune diseases, genetic predisposition to MG probably involves multiple genes. Ample evidence suggests that genes within the major histocompatibility complex (MHC) are involved in susceptibility to autoimmune diseases. Both data from the literature and our findings indicate that different genes within the MHC could predispose to various forms of MG, and that particularly the tumour necrosis factor genes may play a role in the association between the different thymic disorders and MG.

Reduction of the DM-associated homeo domain protein (DMAHP) mRNA in different brain areas of myotonic dystrophy patients.

Myotonic dystrophy (DM) is a multisystemic disease caused by expansion of a CTG trinucleotide repeat in the 3' untranslated region of the DMPK protein kinase gene on chromosome 19q13.3. The mechanism by which this expansion causes disease remains unknown. It has been suggested that CTG expansion not only affects the expression of the DMPK gene, but also alters the nuclear RNA metabolism and expression of neighboring genes. DMAHP, which is expressed in various human tissues, including skeletal muscle, heart and brain, is immediately distal to the 3' end of DMPK gene, in a CpG island which contains the CTG repeat. Here we report a 4- to 5-fold reduction of the expression of the DMAHP gene in different brain areas of DM patients. Our results demonstrate that [CTG]n expansion alters the brain DMAHP mRNA expression supporting a dominant-negative effect at the cellular level of DM [CTG]n mutation. The reduced brain expression of DMAHP could explain cerebral impairment in DM patients.

Effect of leuprorelin acetate on cell growth and prostate-specific antigen gene expression in human prostatic cancer cells.

OBJECTIVES: We investigated modulation of cell growth and prostate-specific antigen (PSA) gene expression in prostatic cancer cells by the luteinizing hormone-releasing hormone analog (LH-RHa), leuprorelin acetate, alone or combined with other agents. METHODS: The effect of the analog on proliferation of both androgen-sensitive and -insensitive prostate cancer cells, maintained in different culture conditions, was evaluated by cell counts at various intervals of time. Basal expression of PSA gene and its variations were determined by a reverse transcriptase-polymerase chain reaction assay. RESULTS: LH-RHa is ineffective in regulating cell growth, when used alone in both hormone-sensitive and -insensitive cell lines. Nevertheless, it counteracts the stimulatory action of androgens on proliferation of LNCaP cells, which respond to low concentrations of dihydrotestosterone. Moreover, LH-RHa has an inhibitory effect on the mitogenic action of epidermal growth factor (EGF) in androgen-unresponsive PC-3 cells. The analog reduces PSA gene expression in both hormone-sensitive and -insensitive cells. Interestingly, it counteracts the gene expression induced by androgens in LNCaP cells and by EGF in PC-3 cells. CONCLUSIONS: These data show that LH-RHa may behave like a negative growth factor, which directly regulates cell growth and PSA gene expression. Moreover, our findings support the idea that growth factors may interfere with the androgen signalling pathway.

Effect of Leuprorelin Acetate on Cell Growth and Prostate-Specific Antigen Gene Expression in Human Prostatic Cancer Cells.

Objectives: We investigated modulation of cell growth and prostate-specific antigen (PSA) gene expression in prostatic cancer cells by the luteinizing hormone-releasing hormone analog (LH-RHa), leuprorelin acetate, alone or combined with other agents. Methods: The effect of the analog on proliferation of both androgen-sensitive and -insensitive prostate cancer cells, maintained in different culture conditions, was evaluated by cell counts at various intervals of time. Basal expression of PSA gene and its variations were determined by a reverse transcriptase-polymerase chain reaction assay. Results: LH-RHa is ineffective in regulating cell growth, when used alone in both hormone-sensitive and -insensitive cell lines. Nevertheless, it counteracts the stimulatory action of androgens on proliferation of LNCaP cells, which respond to low concentrations of dihydrotestosterone. Moreover, LH-RHa has an inhibitory effect on the mitogenic action of epidermal growth factor (EGF) in androgen-unresponsive PC-3 cells. The analog reduces PSA gene expression in both hormone-sensitive and -insensitive cells. Interestingly, it counteracts the gene expression induced by androgens in LNCaP cells and by EGF in PC-3 cells. Conclusions: These data show that LH-RHa may behave like a negative growth factor, which directly regulates cell growth and PSA gene expression. Moreover, our findings support the idea that growth factors may interfere with the androgen signalling pathway.

Tumour necrosis factor beta gene polymorphisms in myasthenia gravis.

Genetic analyses indicate that genes within the major histocompatibility complex (MHC) can be involved in susceptibility to autoimmune disease. To investigate the role of the tumour necrosis factor beta (TNFB) gene in myasthenia gravis (MG) susceptibility, we analysed an NcoI polymorphism within the TNFB gene in 63 MG patients and 93 healthy individuals. When patients were subdivided according to thymic pathology, we found differences between MG patients with thymic hyperplasia and thymoma versus controls. In MG patients with thymic hyperplasia we found a positive association with the TNFB*1 allele [Relative risk (RR): 2.6; P < 0.001] and phenotype (RR: 1.8; P < 0.005) and a negative association with the TNFB*2/2 genotype (RR: 0.2; P < 0.001) when compared to the controls. On the other hand, in MG patients with thymoma we found a positive association with the TNFB*2/2 genotype (RR: 5.6; P < 0.01) and a negative association with the TNFB*1 allele (RR: 0.3; P < 0.05) and *1/2 genotype (RR: 0.2; P < 0.01). These data suggest that the two different forms of MG can have different pathogenesis and that the TNFB gene could influence susceptibility to MG.

[Hormone receptors and growth factors in carcinoma of the prostate]. / Recettori ormonali e fattori di crescita nel carcinoma della prostata.

The therapeutic and prognostic significance of androgen receptors in prostatic carcinoma has been examined on the basis of data obtained with the different techniques used in receptor determination. Moreover, the role of some polypeptide growth factors in the regulation of prostatic cancer growth and progression has been reviewed. Great attention has been focused on in vitro models utilized to investigate androgen receptor alterations and the effects of the different positive and negative regulators of prostatic carcinoma cell growth.

Different expression of the myotonin protein kinase gene in discrete areas of human brain.

We have examined the myotonin protein kinase (MT-PK) gene expression in different human brain areas. Four different spliced forms, comprising exons 13 and 14, were identified and characterized. One form (MYOT-A), lacking the entire exon 13, had not been detected in other studies and it is likely to be a brain-specific transcript. Different brain areas show a specific transcription pattern. These results suggest that MT-PK may have specialized functions in different areas of central nervous system. Alterations of this complex expression pattern could be responsible for the mental status impairment observed in myotonic dystrophy patients.

Familial autoimmune myasthenia gravis: report of four families.

Four families each with two patients with autoimmune myasthenia gravis or related conditions are reported. All clinical forms of myasthenia gravis were represented and different disease types were found within the same family. Either one or two generations could be affected and no association with a single HLA haplotype was found. The frequency of familial autoimmune myasthenia gravis is very low and the genetic factors involved seem to be different from MHC genes.

Failure in detecting mRNA transcripts from the mutated allele in myotonic dystrophy muscle.

The expression of myotonin-protein kinase (MT-PK) gene was studied in myotonic dystrophy (DM) muscle and normal controls using a polymerase chain reaction protocol to analyse the 3' intragenic p(CTG) polymorphism. Unaffected individuals show bi-allelic expression, while the sole wild-type allele was transcribed in DM muscle. Our findings support a gene dosage effect in the pathogenesis of the disease.

Polymerase chain reaction in the detection of mRNA transcripts from the slow skeletal troponin T (TNNT1) gene in myotonic dystrophy and normal muscle.

Recent studies have shown that the gene encoding for the slow skeletal troponin isoform T (TNNT1) is located on the proximal long arm of human chromosome 19 in the myotonic dystrophy (DM) region. In order to test TNNT1 as a candidate gene for DM, we have isolated TNNT1 cDNA from skeletal muscle from two healthy individuals and from two patients with DM. Sequencing of the TNNT1 cDNA from the DM and normal muscle revealed two sequence variants but no transcriptionally significant mutations. This work rules out a defect in the coding segment of TNNT1 as a cause of DM and provides a polymerase chain reaction protocol for studying troponin T gene expression.

Accuracy of reinnervation by peripheral nerve axons regenerating across a 10-mm gap within an impermeable chamber.

The axon regeneration following a peripheral nerve injury often fails to restore a complete functional recovery. One of the causes of this unsatisfactory result has been attributed to regrowth of regenerating fibers to inappropriate peripheral targets. The accuracy of reinnervation by axons regenerating across a 10-mm gap within an impermeable chamber has been studied by using a sequential retrograde double-labeling technique. Despite the long gap between the nerve stumps, at 4 weeks a mean of 30.5% of the regenerating axons can reinnervate the original muscular area. These data confirm previous studies in which a preferential reinnervation is reported not to be absolutely dependent on the axon's mechanical alignment.

Identity between cytoplasmic and membrane-bound S-100 proteins purified from bovine and rat brain.

Cytoplasmic and membrane-bound S-100 proteins were purified to homogeneity from bovine and rat brain. Cytoplasmic and membrane-bound S-100 from single species are identical by immunological, electrophoretic, spectrophotometric, and functional criteria. Cytoplasmic and membrane-bound S-100 from bovine brain consists of nearly equal amounts of S-100a and S-100b, whereas cytoplasmic and membrane-bound S-100 from rat brain consists mostly of S-100b. The functional role of membrane-bound S-100 remains to be elucidated.