Short lingual frenum in infants, children and adolescents. Part 2: Lingual frenum release. Functional surgical approach.

AIM: The aim of this study was to review the craniofacial growth impairment and different malfunctions associated with short lingual frenum and to assess the validity of lingual frenum surgery based on minimally invasive laser release with a myofunctional approach. MATERIALS AND METHODS: Thirty patients, children and adolescents whose ages ranged from 8 years to 18 years, diagnosed with a short lingual frenum and concomitant orthodontic problems and/or presence of associated muscular or postural problems, were treated in this study. Pre-operative tongue assessment was performed following morphological and functional criteria, consisting of measurement of the free tongue, and of visual assessment of tongue protrusion out of the mouth and elevation to the incisive palatal papilla. Postural evaluation was assessed in frontal and lateral view. Laser surgery was completed with local anaesthesia, using Erbium YAG laser (2940 nm, LightWalker, AT-Fotona, Ljubljana, Slovenia) equipped with sapphire conical tip (600 micron), with energy ranging from 120 to 160 mJ, at 15 Hz frequency, and varying the adjustable pulse duration from 300 µs to 600 µs. RESULTS: Significant improvement was noted in 29 of 30 patients comparing preoperative scores to both three-week and two-month post-op scores. Postural improvement was found in 18 of 30 patients, indicating the multifactorial involvement of different causes for correct body posture. CONCLUSION: This study confirmed the validity of Erbium:YAG laser surgery as an effective technique in children and adolescents to release a short lingual frenum. The functional approach of the procedure performed with the Erbium:YAG laser, and the concomitant myofunctional therapy demonstrated to be simple and safe in children, and adolescents. Because of the multifactorial causes involved in correct body posture, an adequate osteopathic therapy is important to successfully complete the full body rehabilitation.

Laser labial frenectomy: a simplified and predictable technique. Retrospective clinical study.

AIM: Anomalous maxillary median labial frenum may be associated with undesired effects such as persistence of diastema between anterior teeth or traction of marginal gingiva. The aim of this study was to propose a surgical frenum repositioning technique that is minimally invasive, safe, easy, reproducible, and predictable. Another objective of the study was to identify clinical scenarios that could have indication for labial frenectomy associated with early orthodontic therapy, so as to justify early frenum repositioning in children. A retrospective assessment of clinical outcomes of this technique is described. MATERIALS AND METHODS: A total of 20 frenectomies were performed on children aged 8 to 10 years. Frenectomies were performed with Er:YAG laser set at 150mJ 2.25-3.0W and 15-20 pulse per second, with water spray. Recall visits were done at 7, 21 and 90 days and 1, 2, 3 and 4 years. RESULTS: At post-operative visits, all patients reported no post-operative pain or minimal discomfort. None experienced post-operative bleeding at a distance of few hours. All patients reported that the procedure was well tolerated and "acceptable". No recurrences occurred 4 years after frenectomy. CONCLUSION: The Er:YAG laser used in this study allowed considerable reduction of the operating time, reducing the amount of local anaesthetic used as well as avoiding surgical sutures. The surgical design and technique also minimised post-operative discomfort and complications resulting in stable healing overtime, making the procedure fully accepted by children.

Paediatric laser dentistry. Part 2: Hard tissue laser applications.

AIM: Erbium lasers can provide effective and minimally invasive caries removal in children. The bonding phase remains a critic step as well as the choice of material. Glass ionomers exhibits lower bonding properties in laser irradiated teeth compared to the conventional method or to composite and resin modified glass ionomer. Laser can also provide effective decontamination and coagulation effects in vital and non vital pulp therapy of primary teeth, improving and simplifying the cleaning and disinfecting steps.

Paediatric laser dentistry. Part 4: Soft tissue laser applications.

AIM: Lasers can provide effective soft tissues applications in children. All the wavelengths produce incision and vaporisation of oral tissues, together with a high bactericidal effect. The haemosthatic effect varys according to the wavelength used, and the choice of a visibile, near, medium or far infrared laser allows a better interaction with specific targets, gingiva, mucosa, frenum, or oral pathology.

Lingual frenectomy: functional evaluation and new therapeutical approach.

AIM: When ankyloglossia is relatively severe and generates mechanical limitations and functional challenges, surgical reduction of the frenum is indicated. MATERIALS AND METHODS: Laser technique is an innovative, safe and effective therapy for frenectomy in both children and adolescents. Erbium:YAG laser (2940nm) can be useful for paediatric dentist: 1.5W at 20pps is a commonly used average power to easily, safely and quickly cut the frenum. RESULTS: Usually after laser frenectomy, the postoperative symptoms and relapse are absent. CONCLUSION: Early intervention is advisable to reduce the onset of alterations correlated to the ankyloglossia. A multidisciplinary approach to the problem is advisable, in collaboration with orthodontist, physiotherapist and speech therapist, to better resolve the problem.

Prognostic value of CCND1 gene status in sporadic breast tumours, as determined by real-time quantitative PCR assays.

The CCND1 gene, a key cell-cycle regulator, is often altered in breast cancer, but the mechanisms underlying CCND1 dysregulation and the clinical significance of CCND1 status are unclear. We used real-time quantitative PCR and RT-PCR assays based on fluorescent TaqMan methodology to quantify CCND1 gene amplification and expression in a large series of breast tumours. CCND1 overexpression was observed in 44 (32.8%) of 134 breast tumour RNAs, ranging from 3.3 to 43.7 times the level in normal breast tissues, and correlated significantly with positive oestrogen receptor status (P=0.0003). CCND1 overexpression requires oestrogen receptor integrity and is exacerbated by amplification at 11q13 (the site of the CCND1 gene), owing to an additional gene dosage effect. Our results challenge CCND1 gene as the main 11q13 amplicon selector. The relapse-free survival time of patients with CCND1-amplified tumours was shorter than that of patients without CCND1 alterations, while that of patients with CCND1-unamplified-overexpressed tumours was longer (P=0.011). Only the good prognostic significance of CCND1-unamplified-overexpression status persisted in Cox multivariate regression analysis. This study confirms that CCND1 is an ER-responsive or ER-coactivator gene in breast cancer, and points to the CCND1 gene as a putative molecular marker predictive of hormone responsiveness in breast cancer. Moreover, CCND1 amplification status dichotomizes the CCND1-overexpressing tumors into two groups with opposite outcomes.

hTERT expression in sporadic renal cell carcinomas.

Human telomerase is a specialized reverse transcriptase that catalyses telomeric repeat addition at the ends of chromosomes. Activation of this enzyme is one of the key steps in cell immortalization and carcinogenesis, and one of its components, hTERT, is considered as the rate-limiting factor. While telomerase activity was found to be prognostically relevant in various cancers, results obtained from renal cell carcinomas (RCC) failed to show any correlation with the usual prognostic factors. The aim of the study was to reassess the role of telomerase and its hTERT component in the biological behaviour of RCC using new quantitative techniques, such as the quantitative evaluation of hTERT mRNA level by a real-time RT-PCR procedure and the measuring of telomerase activity by an ELISA TRAP assay. Since experimental evidence supports a relationship between cell proliferation or c-myc expression and telomerase, the proliferation index and c-myc mRNA levels were also studied. Forty-one RCC (29 conventional renal cell carcinomas (CRCC), 10 papillary RCC and two urothelial carcinomas) were studied. In 73% of cases, normalized hTERT mRNA expression was significantly higher in the tumour sample than in the normal tissue. Telomerase activity was detected in 63% of RCC, while corresponding normal tissue was always negative. Analysis of correlations showed firstly that both telomerase activity and hTERT mRNA level were lower in the group of CRCC versus non-CRCC (TRAP: 0.3+/-0.1 versus 0.6+/-0.2, p<0.05; hTERT/PO mRNA: 5+/-3 versus 37+/-8, p<0.001, respectively); secondly, that in the group of CRCC, hTERT mRNA expression level was correlated with the stage of the tumour (p=0.01); and thirdly, that no correlation was observed between c-myc mRNA level and hTERT mRNA level. In conclusion, these results support the involvement of telomerase in RCC and the potential interest of hTERT mRNA quantification.

Quantification of TEL-AML1 transcript for minimal residual disease assessment in childhood acute lymphoblastic leukaemia.

Strategies currently used for residual disease detection in acute lymphoblastic leukaemia (ALL) rely on polymerase chain reaction (PCR) detection of immunoglobulin and T-cell receptor rearrangements. The TEL-AML1 fusion transcript, which is associated with t(12;21) (p13;q22), is found in 25% of childhood B-cell precursor ALL, and represents an interesting alternative target. We compared two methods for quantitating TEL-AML1 fusion transcripts: competitive PCR and real-time PCR. These techniques showed similar sensitivity (5 x 10(-5)) and reproducibility. Giving highly correlated results, both techniques can be conveniently used for TEL-AML1 transcript quantification. The constancy of TEL-AML1 expression was evaluated by measuring TEL-AML1 transcripts at different steps of the cell cycle, and in 21 cases of ALL at diagnosis. No major variation in TEL-AML1 expression was observed during the cell cycle or in 20/21 of the ALL patients. Residual disease was then determined after completion of induction therapy in 20 patients with a TEL-AML1-positive ALL. Seven patients out of 20 (35%) were still positive, including two patients with high level of residual blasts (close to or beyond 10(-2)). When comparison was possible, results obtained using TEL-AML1 quantification were in accordance with those obtained using T-cell receptor rearrangements analysis.

Real-time PCR as a new tool for quantifying Leishmania infantum in liver in infected mice.

The parasitic loads of mouse livers experimentally infected with Leishmania infantum were determined using a double real-time quantitative PCR test targeted to the parasite DNA polymerase gene and to the mouse brain-derived neutrophic factor gene. The Leishmania DNA copy number was normalized to the number of mouse gene copies in order to quantify the former independently of liver weight. The correlation coefficient with the microtitration method was 0.66. This PCR assay can be considered for experimental pharmaceutical studies.

Analysis of the human CGB/LHB gene cluster in breast tumors by real-time quantitative RT-PCR assays.

The six genes of the human chorionic gonadotropin beta subunit (CGB) and the gene of the luteinizing hormone beta subunit (LHB) are located in a cluster that spans 50 kb on chromosome 19q13.3. Only genes CGB7, B8, B5 and B3 can generate the human chorionic gonadotropin (hCG) beta molecule. The other two genes, CGB1 and B2, encode unidentified proteins. We have previously shown that malignant breast transformation is associated with the emergence of the 'trophoblastic' CGB genes (B8, B5 and B3), in addition to the CGB7 gene, which is the only CGB gene expressed in normal breast tissue. To better understand the dysregulation of the CGB/LHB gene cluster in breast cancer, we have developed real-time quantitative RT-PCR assays to analyze each subgroup of genes (the overall CGB genes, CGB1 and B2 together, and LHB alone) in 17 unilateral invasive primary breast tumor RNAs. We also analyzed the chorionic gonadotropin alpha (CGA) gene coding for the human CGA subunit. We found that the emergence of the 'trophoblastic' CGB genes in breast tumors is (i) accompanied by an increase in the total CGB mRNA steady-state level, (ii) mainly due to overexpression of genes CGB8, B5 and B3 (expression of other genes in the CGB/LHB gene cluster (CGB7, B2, B1 and LHB) changes little if at all), and (iii) not accompanied by overexpression of the CGA gene which is necessary to produce ectopic hCG heterodimeric hormone in breast tumor cells, these latter which yet expressed the LH/CG receptor. These observations suggest that it is mainly the CGB8, B5 and B3 genes which are upregulated in the 19q13.3 CGB gene cluster in breast tumors. They also point to a role (like growth factor) of the CGbeta subunit in breast tumorigenesis, via a novel pathway independent of the LH/CG receptor.

Identification of CGA as a novel estrogen receptor-responsive gene in breast cancer: an outstanding candidate marker to predict the response to endocrine therapy.

The estrogen receptor (ER) status of breast tumors is used to identify patients who may respond to endocrine agents such as tamoxifen. However, ER status alone is not perfectly predictive, and there is a pressing need for more reliable markers of endocrine responsiveness. Here, we identified the well-known CGA gene (coding for the alpha subunit of glycoprotein hormones) as a new ERalpha-responsive gene in human breast cancer cells. We used a real-time quantitative reverse transcription-PCR assay to quantify CGA mRNA copy numbers in a large series of breast tumors. CGA overexpression (> 10 SD above the mean for normal breast tissues) was observed in 44 of 131 (33.6%) breast tumor RNAs, ranging from 20 to 16,500 times the level in normal breast tissues; the highest levels of CGA gene expression were close to those observed in placenta. Significant links were observed between CGA gene overexpression and Scarff-Bloom-Richardson histopathological grade I+II (P = 0.015), and progesterone (P = 0.0009) and estrogen (P < 10(-7)) receptor positivity, which suggested that CGA is a marker of low tumor aggressiveness. We observed CGA mRNA overexpression in 44 of 90 (48.9%) ERalpha-positive tumors and in none of the 41 ERalpha-negative tumors. Immunohistochemical studies demonstrated that human chorionic gonadotropin alpha protein was strictly limited to ERalpha-positive tumor cells. Overexpression of the CGA gene was not accompanied by overexpression of the CGB gene. Our results also suggest that CGA could be a more reliable marker than PS2 and PR for ERalpha functionality and, thus, for endocrine responsiveness. Moreover, the CGA marker has the added value of dichotomizing ERalpha-positive patients into two subgroups of similar size. Specific antibodies directed to secreted human chorionic gonadotropin alpha protein are commercially available, thus facilitating the future application of this marker to the clinical management of breast cancer.

Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum.

Several PCR assays have been developed for detecting Aspergillus fumigatus DNA in blood of patients with invasive aspergillosis. However, the best blood fraction to be assayed has not been defined and the multicopy genes used as the DNA targets for amplification not characterized. Firstly, we developed a real-time PCR assays based on the TaqMan technology targeted to a single copy gene. To compare serum, white cell pellet, and plasma for effectiveness as blood assay fractions, we spiked whole blood with A. fumigatus DNA and processed these fractions similarly. The difference between white cell pellet and serum was not significant. In contrast, the yield from plasma was 10 times lower than from serum. Then, we compared serum processed immediately or after 24 h at room temperature and observed a lower yield after 24 h. Secondly, a real-time PCR assay targeted to a mitochondrial gene was also developed. The copy number was estimated between 9 and 10 mitochondrial genes per single copy gene. Therefore, we recommend serum, stored and frozen as soon as possible, to be used for detecting circulating A. fumigatus DNA for diagnosis. Moreover, the mitochondrial multicopy gene was characterized in order to compare results from different patients.

Human TIP49b/RUVBL2 gene: genomic structure, expression pattern, physical link to the human CGB/LHB gene cluster on chromosome 19q13.3.

Bacterial DNA helicase RuvB protein is an essential component in homologous recombination and DNA double-strand break repair. Here, we report the gene structure of TIP49b/RUVBL2, a second putative human homologue of the bacterial RuvB gene. This gene contains 15 exons and 14 introns. The TIP49b/RUVBL2 open reading frame encodes a protein of 463 amino acids, showing 43% identity with the RUVBL1 protein. The TIP49b/RUVBL2 gene is physically linked to the human CGB/LHB gene cluster on chromosome 19q13.3. Genomic sequence analysis revealed that the TIP49b/RUVBL2 gene is very close (55 nucleotides in length) to the LHB gene, in the opposite orientation. The very close co-location of the mouse homologues of the human TIP49b/RUVBL2 and LHB genes was also conserved on mouse chromosome 7. Co-ordinated transcriptional regulation between the TIP49b/RUVBL2 and LHB genes was not observed. TIP49b/RUVBL2, like RUVBL1, was expressed ubiquitously in all human tissues examined and more strongly in testis. As TIP49b/RUVBL2 is expected to be involved in recombination repair and is located in a chromosome region frequently amplified in breast cancer, we quantified TIP49b/RUVBL2 gene expression by using real-time quantitative RT-PCR in a series of breast tumour samples. None of the tumour samples showed an altered TIP49b/RUVBL2 transcription level relative to normal breast tissue.

Quantitation of hTERT gene expression in sporadic breast tumors with a real-time reverse transcription-polymerase chain reaction assay.

Recent observations support the notion that telomerase expression is essential for the formation of human tumor cells [W-C. Hahn et al., Nature (Lond.), 400: 464-468, 1999]. The expression pattern of hTERT, the human telomerase catalytic subunit gene, is a rate-limiting determinant of the enzymatic activity of human telomerase. We have developed a real-time quantitative RT-PCR assay based on Taq-Man fluorescence methodology to quantify the full range of hTERT mRNA copy numbers. We validated the method on a series of 134 unilateral invasive primary breast cancer patients with known long-term outcome. Three-quarters of the breast tumors (75.4%; 101 of 134) were hTERT positive, i.e., contained detectable and quantifiable hTERT mRNA. hTERT-positive patients had significantly shorter relapse-free survival (P = 0.017) after surgery compared with hTERT-negative patients. The prognostic significance of hTERT status persisted in Cox multivariate regression analysis. When we subdivided hTERT-positive patients (n = 101) into three equal groups (tumors showing small, intermediate, or high increase in hTERT mRNA content), we observed statistical (or a trend toward) links between high hTERT mRNA levels and Scarff-Bloom-Richardson histopathological grade III (P = 0.066), and negative estrogen (P = 0.002) and progesterone (P = 0.048) receptor status, and therefore with higher aggressiveness of breast tumors. High hTERT mRNA levels were also linked to MYC gene overexpression (P = 0.007). These findings show that the quantitative evaluation of hTERT mRNA can have important prognostic significance in human breast cancer. In addition, our simple, rapid, and semiautomated assay method is suitable for routine hTERT mRNA detection and quantification and will be a powerful tool in large, randomized, prospective, cooperative group trials and in the hTERT-based therapy project.

A short induction regimen of interferon-alpha is not effective for treatment of relapse in chronic hepatitis C: a randomized trial. For the multicentre GER-CYT-01 group.

The aim of this work was to assess the effect of a high-dose (10 million units, MU) short-duration (14 weeks) interferon-alpha2b (IFN-alpha2b) regimen in relapsers compared with the standard IFN regimen of 3 MU three times weekly (t.i.w.) for 6 months. Fifty-eight non-cirrhotic patients (who had relapsed after previous treatment with IFN) with chronic hepatitis were randomized: 29 to the high-dose, short-duration regimen and 29 to the standard regimen. By the end of IFN therapy, in the high-dose, short-duration group alanine aminotransferase (ALT) normalization was observed in 23 (79%) of 29 patients, and undetectable hepatitis C virus (HCV) RNA in eight (28%) vs 25 (86%) and 11 (38%) of the 29 patients in the standard group, respectively (P = NS). At the end of the 72-week follow-up, in the high-dose, short-duration group a sustained ALT normalization was observed in two (7%) patients, and undetectable HCV RNA in 0 (0%) vs five (17%) and four (14%) patients in the standard group (P = NS). There was less fibrosis improvement in the high-dose, short-duration group (two of 26 patients, 8%) than in the standard group (eight of 25 patients, 32%) (P = 0.04). Tolerance to IFN was good and similar in the two groups. In conclusion, in IFN relapsers, high-dose, short-duration treatment with IFN-alpha has no advantage when compared to a 6-month treatment with 3 MU IFN t.i.w.

Extrahepatic manifestations of chronic hepatitis C. MULTIVIRC Group. Multidepartment Virus C.

OBJECTIVE: To assess the prevalence of clinical and biologic extrahepatic manifestations of hepatitis C virus (HCV) infection and to identify associations between clinical and biologic manifestations. METHODS: To analyze the natural history of extrahepatic manifestations of HCV infection, we reviewed only the data recorded prospectively during the first visit of 1,614 patients with chronic HCV infection, coming from a single monocenter cohort. Exclusion criteria were positivity for hepatitis B surface antigen or human immunodeficiency virus. The prevalence of dermatologic, rheumatologic, neurologic, and nephrologic manifestations; diabetes; arterial hypertension; autoantibodies; and cryoglobulins were assessed. Then, using multivariate analysis, we identified demographic, biochemical, immunologic, virologic, and liver histologic factors associated with the presence of extrahepatic manifestations. RESULTS: At least 1 clinical extrahepatic manifestation was observed in each of 1,202 patients (74%). Five manifestations had a prevalence >10%: arthralgia (23%), paresthesia (17%), myalgia (15%), pruritus (15%), and sicca syndrome (11%). Four biologic abnormalities had a prevalence >5%: cryoglobulins (40%), antinuclear antibodies (10%), low thyroxine level (10%), and anti-smooth muscle antibodies (7%). Only vasculitis, arterial hypertension, purpura, lichen planus, arthralgia, and low thyroxine level were associated with cryoglobulin positivity. By univariate and multivariate analyses, the most frequent risk factors for the presence of clinical and biologic extrahepatic manifestations were age, female sex, and extensive liver fibrosis. CONCLUSION: Extrahepatic clinical manifestations are frequently observed in HCV patients and involve primarily the joints, muscles, and skin. The most frequent immunologic abnormalities include mixed cryoglobulins, antinuclear antibodies, and anti-smooth muscle antibodies. The most frequent risk factors for the presence of clinical and biologic extrahepatic manifestations are advanced age, female sex, and extensive liver fibrosis.

Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications.

BACKGROUND: Gene amplification/overexpression of ERBB2 (HER2, neu) is a major event in human breast tumorigenesis. ERBB2-based therapeutic agents and ERBB2-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen breast cancer patients for ERBB2 alterations. METHODS: We have developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify ERBB2 gene expression at the mRNA level in breast tumors. This recently developed method of nucleic acid quantification in homogeneous solutions has the potential for a wide dynamic range, interlaboratory agreement, and high-throughput capacity without tedious post-PCR processing. The ERBB2 mRNA signal was normalized to the signal for TATA box-binding protein mRNA. RESULTS: The dynamic range was >1000-fold. The relationship between C(t) and log starting concentration was linear (r(2) >/=0.99). The mean (SD) normalized expression of ERBB2 in healthy breast tissue was 0.95 (0.37). Overexpression (>5 SD above mean for healthy breast) of the ERBB2 gene was observed (at 3.2- to 135-fold) in 23 (17%) of 134 breast tumor RNA samples. As expected, ERBB2 overexpression was present in all tumors with ERBB2 gene amplification but was uncommon and at a low ratio (<5) in breast cancers without gene amplification. CONCLUSIONS: This new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.

TaqMan PCR-based gene dosage assay for predictive testing in individuals from a cancer family with INK4 locus haploinsufficiency.

BACKGROUND: A genetic syndrome of cutaneous malignant melanoma and nervous system tumors recently has been characterized and shown to be linked to the INK4 locus in the 9p21 region. Hemizygosity at adjacent physically mapped microsatellite markers indicated deletion of p16, p19, and p15 clustered tumor suppressors. Because individuals from this family could benefit from predictive testing in terms of cancer prevention, we developed a direct test without need to analyze parental DNAs to comply with the rules of individual consent and secrecy. METHODS: We developed an assay using TaqManTM real-time quantitative PCR, with p15 as the test sequence and albumin (ALB) as the reference gene. The normalized ratio of p15/ALB is expected to yield a value of approximately 1 in individuals without the deletion, whereas a ratio of approximately 0.5, indicating p15 haploinsufficiency, is expected in predisposed individuals. RESULTS: All patients harboring the previously defined at-risk haplotype were correctly identified using this approach. In six individuals with deletions, the p15/ALB ratios were 0.472-0.556 (SD, 0.013-0.078). In the five individuals without deletions, the ratios were 0.919-1.019 (SD, 0.006-0.075). CONCLUSIONS: This is the first report of a high-throughput, automatable gene dosage assay successfully applied to the identification of a germ-line deletion. This approach, not limited by marker informativeness or the need for harvesting live cells, can be applied to any condition with haploinsufficiency and extended to the characterization of most abnormalities of the ploidy.