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.

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.

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.

Quantitation of MYC gene expression in sporadic breast tumors with a real-time reverse transcription-PCR assay.

MYC gene overexpression was identified recently as a downstream step at the end of the Wnt/APC/beta-catenin pathway dysregulation observed in colorectal cancer (T-C. He et al., Science (Washington DC), 281: 1509-1512, 1998). It thus appears that an excess of c-myc protein is a primary cause of numerous cancers. In breast cancer, MYC has been studied mostly at the DNA level because of the poor quality of available antibodies against the protein product. The renewed interest in MYC calls for a sensitive and accurate method for analyzing MYC overexpression in breast tumors. We have developed a real-time quantitative reverse transcription-PCR assay based on TaqMan fluorescence methodology to quantify the MYC mRNA copy number. We validated the method on a large series of breast tumors. MYC gene overexpression was observed in 29 of 134 (22%) breast tumor RNAs, ranging from 3.2 to 19 times the level in normal breast tissues. These data imply that dysregulated MYC gene expression is potentially involved in the pathogenesis of breast cancer, especially by favoring local cell proliferation. We also found that MYC gene overexpression was rarely due to an increased MYC gene copy number in breast cancer. This new, simple, rapid, and semiautomated method will be useful for screening cancer patients for MYC overexpression and will prove a powerful tool in large, randomized, prospective, cooperative group trials and in the MYC-based therapy project.

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.

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.

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.

Establishment of T-cell lines from infiltrated NOD islets by stimulation of specific T-cell receptor V beta segments.

The aims of this study were firstly to establish permanent T-cell lines from infiltrated NOD islets, by repeated stimulation of the antigen T-cell receptor with anti-V beta monoclonal antibodies (mAb) and, secondly, to characterize some of their cytotoxic and pathogenic properties. The use of anti-V beta antibodies was aimed at driving the expansion of the T cells in the absence of pancreatic antigen and, at the same time, at selecting lymphocytes expressing a given V beta gene product as an element of their TCR. Twelve lines were established as long-term cultures by regular stimulation with plastic-bound anti-V beta 6 or anti-V beta 8 mAb. The eight lines cultured with anti-V beta 6 mAb were phenotyped as early as one month after initiation and were all V beta 6+/V beta 8-. Three were CD8+ and five CD4+. Of the four lines established with anti-V beta 8 mAb, three were V beta 8+/V beta 6- and one (FD) was unexpectedly phenotyped as V beta 6+/V beta 8-. Clones derived from the FD line confirmed the expression of V beta 6. The cell-mediated cytolytic properties of the 12 lines were evaluated in two independent assays: an antibody-redirected assay to measure the lytic potential irrespective of antigen specificity and a direct cytolytic assay on YAC cells for assessing NK-like activity. The results indicate that practically all the lines (11 out of 12), irrespective of their CD4/CD8 phenotype or V beta expression, can exert cell-mediated cytotoxicity when their TCR/CD3 complex is linked to a target cell. On the other hand, anti-YAC activity is almost exclusively confined to CD8+ cell lines. Pathogenicity was evaluated in two CD4+ T cell lines, one which showed cytolytic activity in the redirected assay but not in the YAC assay (FD) and one which was totally devoid of cytotoxic activity (AH). The two lines were injected into newborn NOD mice with or without CD8+ polyclonal T cells. The results indicate that FD, the cytotoxic line, can induce severe lesions of insulitis when coinjected with polyclonal CD8+ T cells. In contrast, AH, the non-cytotoxic line, injected under the same conditions, induces no lesions. Altogether, the present data demonstrate the feasibility of establishing permanent T-cell lines on the basis of V beta expression.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer.

Gene amplification is a common event in the progression of human cancers, and amplified oncogenes have been shown to have diagnostic, prognostic and therapeutic relevance. A kinetic quantitative polymerase-chain-reaction (PCR) method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real-time, was used to quantify gene amplification in tumor DNA. Reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components is limited during the exponential phase, meaning that values are highly reproducible in reactions starting with the same copy number. This greatly improves the precision of DNA quantification. Moreover, real-time PCR does not require post-PCR sample handling, thereby preventing potential PCR-product carry-over contamination; it possesses a wide dynamic range of quantification and results in much faster and higher sample throughput. The real-time PCR method, was used to develop and validate a simple and rapid assay for the detection and quantification of the 3 most frequently amplified genes (myc, ccndl and erbB2) in breast tumors. Extra copies of myc, ccndl and erbB2 were observed in 10, 23 and 15%, respectively, of 108 breast-tumor DNA; the largest observed numbers of gene copies were 4.6, 18.6 and 15.1, respectively. These results correlated well with those of Southern blotting. The use of this new semi-automated technique will make molecular analysis of human cancers simpler and more reliable, and should find broad applications in clinical and research settings.

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.

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.

Modeling the impact of interferon alfa treatment on liver fibrosis progression in chronic hepatitis C: a dynamic view. The Multivirc Group.

BACKGROUND & AIMS: Impact of hepatitis C treatment has never taken into account the dynamics of fibrosis progression. This study assessed the impact of interferon on liver fibrosis progression in patients with chronic hepatitis C according to 3-month aminotransferase activity response. METHODS: We recruited 287 patients, 185 treated and 102 control, with paired biopsy specimens. Before follow-up, the fibrosis progression rate per year was estimated as the ratio between fibrosis stage in METAVIR units (1 U, 1 stage; 4 U, cirrhosis) and the duration of infection. During follow-up, fibrosis progression was assessed by the observed difference between stages divided by duration between biopsies. RESULTS: The median fibrosis progression rate in treated patients decreased compared with the rate before treatment from 0.103 F METAVIR U/yr (95% confidence interval [CI], 0.087-0.120) to 0.000 (95% CI, 0.000-0.000; P

Anchored polymerase chain reaction based analysis of the V beta repertoire in the non-obese diabetic (NOD) mouse.

We have performed extensive analyses of T cell receptor V beta usage in the thymus, the spleen and the infiltrated islets of preclinical non-obese diabetic (NOD) mice. A semiquantitative anchored polymerase chain reaction (An-PCR) protocol has been developed for this purpose. The validity of the method has been first assessed by antibody staining with a panel of anti-V beta monoclonal antibodies (mAb). The results obtained by An-PCR are accurate, reproducible, and in good agreement with cell surface protein staining. A strict comparison between thymus and spleen repertoires reveals no major V beta-specific deletion except the already reported V beta 3 deletion due to Mtv-3. Certain V beta such as V beta 15, 18, 20 are found with a low frequency in the spleen, but the fact that they are also scarce in the thymus probably reflects a poor availability of these genetic elements during beta chain rearrangement rather than negative selection. Other V beta, such as V beta 2, V beta 12 and V beta 14 are significantly more abundant in the spleen than in the thymus. This finding was confirmed by mAb staining for V beta 2 and V beta 14. The expansion asymmetrically affects the CD4+ subset and can be traced back to the mature, single-positive thymocyte subset, suggesting an intrathymic positive selection event. V beta repertoires in infiltrated islets of 13- and 18-week-old, non-diabetic mice are polymorphic. Practically all the V beta found in the peripheral lymphoid tissues are present in the islets, in similar proportions. The major exception is V beta 12, one of the V beta which is subject to expansion during intrathymic differentiation and which is further augmented in the islets, both at 13 and 18 weeks. This increase probably reflects further peripheral amplification of the V beta 12-bearing subset due to encounter with the same ligand as in the thymus or with a cross-reactive motif. Finally, the nucleotide sequencing of all the V beta segments in usage in the NOD strain confirms the absence of allelic polymorphism of V beta-coding regions.