Locally advanced cervical cancer complicating pregnancy: A case of competing risks from the Catholic University of the Sacred Heart in Rome.

A case of stage IB2 cervical cancer at 27 weeks of pregnancy, treated with neoadjuvant chemotherapy followed by radical Cesarean hysterectomy with full pelvic and infra-mesenteric lymphadenectomy, and adjuvant chemo-radiation is described. While she remains without disease, her baby was diagnosed with acute myelogenous leukemia. We highlight the pre-operative work-up, treatment options, safety, feasibility, and outcomes for the mother and her fetus.

Paclitaxel, epirubicin, and cisplatin (TEP) regimen as neoadjuvant treatment in locally advanced cervical cancer: long-term results.

OBJECTIVE: We evaluated the rates of response, operability and long term survival and toxicities in a large series of locally advanced cervical cancer (LACC) patients administered neoadjuvant chemotherapy (NACT) with paclitaxel, epirubicin and cisplatin (TEP) followed by radical surgery (RS). Patients and methods The study included 75 consecutive stages IB2-IVA patients administered NACT with paclitaxel (175mg/m(2)), epirubicin (100mg/m(2)) and cisplatin (100mg/m(2)) on day 1 of a 3-weekly cycle for 2-4cycles. Patients were evaluated for objective response by RECIST criteria and triaged to RS. Progression-free survival (PFS) and overall survival (OS) were calculated from the date of diagnosis to recurrence/progression of disease or death, respectively. RESULTS: Complete and partial clinical response was observed in 13 and 28 patients (56.1% objective responses); radical surgery was amenable in 52 patients (71.2%): 14 patients showed complete/microscopic response to treatment. Overall, recurrence/progression of disease was observed in 36 patients, and all of them experienced death of disease. In the whole series median PFS was 48months (5-year PFS=51.0%), and median OS was 72months (5-year OS=53.0%). Overall, 195 courses were administered; treatment was delayed in 6.7% of patients, while dose reduction was required in 36.5% of patients. Grade 3 leukopenia affected 22 patients (29.7%), while Grades 3 and 4 neutropenia was documented in 17 (22.9%) and 6 (8.1%) patients. In the whole series, we recorded 1 death whose relation with treatment-induced toxicity could not be ruled out. CONCLUSIONS: TEP provided favorable rates of response and operability in LACC patients, and allowed the obtainment of encouraging survival data without carrying out an excessive toxicity.

Phase II study of NGR-hTNF in combination with doxorubicin in relapsed ovarian cancer patients.

BACKGROUND: The NGR-hTNF (asparagine-glycine-arginine-human tumour necrosis factor) is able to promote antitumour immune responses and to improve the intratumoural doxorubicin uptake by selectively damaging tumour blood vessels. METHODS: Patients progressing after ≥ 1 platinum/taxane-based regimen received NGR-hTNF 0.8 µg m(-2) and doxorubicin 60 mg m(-2) every 3 weeks. Primary endpoint was a Response Evaluation Criteria in Solid Tumors-defined response rate with a target of more than 6 out of 37 responding patients. RESULTS: A total of 37 patients with platinum-free interval lower than 6 months (PFI<6; n=25), or between 6 and 12 months (PFI=6-12; n=12) were enrolled. Median baseline peripheral blood lymphocyte count (PBLC) was 1.6 per ml (interquartile range, 1.2-2.1). In all, 18 patients (49%) received more than 6 cycles. Febrile neutropaenia was registered in one patient (3%). Among 35 assessable patients, 8 (23%; 95% CI 12-39%) had partial response (2 with PFI<6; 6 with PFI=6-12) and 15 (43%) had stable disease (10 with PFI<6; 5 with PFI=6-12). Median progression-free survival (PFS) was 5.0 months for all patients, 3.8 months for patients with PFI<6, and 7.8 months for patients with PFI=6-12. Median overall survival (OS) was 17.0 months. Patients with baseline PBLC higher than the first quartile had improved PFS (P=0.01) and OS (P=0.001). CONCLUSION: Tolerability and activity of this combination warrant further randomised testing in patients with PFI<6. The role of PBLC as a blood-based biomarker deserves further investigation.

Prevalence of cervical HPV and attitude towards cervical screening in IBD patients under immunomodulatory treatment: a multidisciplinary management experience.

OBJECTIVE: Therapeutic strategies for Inflammatory Bowel Diseases (IBD: Crohn's disease and Ulcerative Colitis) have improved but the risk for HPV infection in patients under immunomodulatory/biologic treatment is unclear. Objective of the study is to identify the attitude of patients and caregivers to cervical screening. To determine the prevalence of HPV and cervical lesions in IBD patients receiving immunomodulatory/biological treatment. PATIENTS AND METHODS: IBD patients treated with immunomodulators were enrolled from November 2016 to September 2017, thanks to a multidisciplinary cooperation. A survey was administered to enrolled patients as well as to a selected network of IBD expert physicians. Patients who consented underwent gynecological examination, smear, HPV DNA test, colposcopy, vaginal and cervical microbiological swabs. RESULTS: 294 patients from AMICI Onlus Association, 119 patients from the hospital clinic, 30 doctors from national IBD centers participated to the survey. 19 patients from the IBD clinic underwent cervical screening. More than 90% of doctors consider their patients at risk of cervical cancer. A low prevalence of high-risk genotypes and related HPV lesions and an increased prevalence of bacterial vaginosis emerged in the studied population. CONCLUSIONS: Biological drugs could lead to a positive immunomodulation towards HPV infection. In IBD patients an alteration of the vaginal and intestinal microbiota seems to be coexisting.

Significance of epidermal growth factor receptor in advanced ovarian cancer.

PURPOSE: The purpose of this study was to investigate the significance of epidermal growth factor receptor (EGF-R) expression in a group of advanced ovarian carcinomas. PATIENTS AND METHODS: The study was conducted on 72 previously untreated patients with International Federation of Gynecology and Obstetrics (FIGO) stage III-IV disease. The median follow-up was 24 months (range, 4 to 75 months). EGF-R was measured by a radioreceptorial assay. A cutoff of 1.5 fmol per milligram of protein was chosen to define EGF-R positivity. Medians and life tables obtained with the Kaplan and Meier method were analyzed by the log-rank test. The risk of progression was estimated by Cox's proportional hazards model. RESULTS: EGF-R was detected in 54% of primary tumors. When EGF-R was analyzed in different tissue specimens of the same tumor, consistent findings were noted in 88% (seven of eight) of cases. A lower concordance rate (nine of 15; 60%) was found between primary tumors and omental metastases, with a tendency toward higher EGF-R levels in the latter. The EGF-R expression did not significantly correlate with age, stage, grading, and residual tumor after primary surgery. In the univariate analysis, stage IV disease, postoperative residual tumor diameter greater than 2 cm, presence of ascites, and EGF-R positivity were found to be significantly associated with a greater risk of disease progression. In the multivariate analysis, only the postoperative residual tumor and the EGF-R expression remained significantly associated with a high risk of progression. CONCLUSION: Data reported here suggest that the presence of EGF-R in advanced ovarian tumor at the time of the primary surgery identifies a subset of patients with a particularly poor prognosis.

Antiproliferative effect of silybin on gynaecological malignancies: synergism with cisplatin and doxorubicin.

The aim of this study was to test the antiproliferative activity of silybin, a flavonoid, on human ovarian and breast cancer cell lines. Since flavonoids are thought to act through Type II oestrogen binding sites (Type II EBS), silybin binding to Type II EBS was also examined. Silybin, used in concentrations from 0.1 to 20 microM, exerted a dose-dependent growth inhibitory effect on OVCA 433, A2780 parental and drug-resistant ovarian cancer cells, and MCF-7 doxorubicin (DOX)-resistant breast cancer cells (IC50 = 4.8-24 microM). Both L and D diastereoisomers of silybin were effective in inhibiting A2780 WT cell growth (IC50 = 14 and 20 microM, respectively). Flow cytometry revealed that silybin decreased the percentage of cells in the S and G2-M phases of the cell cycle with a concomitant increase in cells in the G0-G1 phase. Silybin was able to compete with [3H]E2 for nuclear but not cytosolic Type II EBS. Its affinity parallels its efficacy in inhibiting cell proliferation. Furthermore, silybin (0.1 and 1 microM) potentiates the effect of cisplatin (CDDP) (0.1-1 micrograms/ml) in inhibiting A2780 WT and CDDP-resistant cell growth. Similar results were obtained on MCF-7 DOX-resistant cells when silybin (0.1 microM) was associated with doxorubicin (0.1-10 micrograms/ml). As assessed by the Berembaum isobole method, the effect of silybin-CDDP and silybin-DOX combinations results in a synergistic action. Using the 'stem cell assay' described by Hamburger and Salmon [Science 1977, 197, 461-463], we found that silybin exerted a dose-dependent inhibition of clonogenic efficiency of cells derived from three ovarian tumours (IC50 = 7.4, 4 and 6.4 microM, respectively). Since CDDP and DOX are the two most commonly used drugs for gynaecological tumours, the clinical application of silybin is currently under investigation in our institute.

Synergistic antiproliferative activity of tamoxifen and cisplatin on primary ovarian tumours.

We looked for the presence of the so-called type II oestrogen binding sites (EBS), in four oestrogen (ER) and progesterone (PR) receptor negative primary ovarian tumours. Moreover, the colony-forming assay was used to evaluate the response of ovarian cancer cells from these primary tumours to tamoxifen and cisplatin used alone or in combination. All tumours contained type II EBS, and tamoxifen was able to compete for [3H] oestradiol binding to these sites. Cisplatin and tamoxifen exhibited a dose-dependent inhibition of colony formation in a range of concentrations between 10 and 1000 micrograms/l and 37 and 3710 micrograms/l, respectively. The combination of the two drugs resulted in a synergistic antiproliferative activity, with a potentiation up to and beyond 50-fold. Our results show that in ovarian cancer tamoxifen interacts with type II EBS with an affinity consistent with the concentration effective both in inhibition of colony formation and in synergising cisplatin activity. Based on the experiments performed the action of tamoxifen on cell growth is independent of ER expression, and could be mediated by type II EBS. The possibility that the association of tamoxifen and cisplatin may result in an improved clinical response in ovarian cancer should be investigated.

Clinical significance of cathepsin D in primary ovarian cancer.

Cathepsin D (Cath D) levels were assayed in a prospective series of 72 patients with primary ovarian carcinoma, by using an immunoradiometric assay. Cath D levels ranged from 2.00 to 45.60 pmol/mg protein with a median value of 15.80 pmol/mg protein. Cath D levels were higher in metastatic deposits than in primary tumors (median 24.12, range 9.33-98.33 pmol/mg protein versus median 12.76, range 2.00-45.20 pmol/mg protein; P = 0.04). The cut-off levels of the lower, median and upper quartiles of the distribution of Cath D were identified to distinguish patients with low, intermediate, and high Cath D content. Cases with low Cath D content showed a lower percentage of complete response to chemotherapy than cases with intermediate and high Cath D content (22% versus 65% and 47%, respectively) (P = 0.003). Moreover cases with high Cath D content showed a worse prognosis with respect to patients with intermediate Cath D levels (P = 0.09). Interestingly, cases with low Cath D content had a shorter progression-free survival with respect to cases with intermediate Cath D content (P = 0.04). Cath D status retained an independent prognostic value when assessed in the multivariate analysis.

N-deacetyl-N-aminoacylthiocolchicine derivatives: synthesis and biological evaluation on MDR-positive and MDR-negative human cancer cell lines.

A new series of N-deacetyl-N-(N-trifluoroacetylaminoacyl)thiocolchicine derivatives 9-15 have been synthesized starting from the corresponding N-deacetylthiocolchicine (3) and the N-trifluoroacetylamino acids 5-8 which were used as a racemic mixture. The trifluoroacetyl protecting group has been removed easily, giving the corresponding N-deacetyl-N-aminoacylthiocolchicines 16-22. Optical pure compounds 9-22 were isolated from the diastereoisomeric mixture or were prepared starting from compound 3 and an optical pure amino acid derivative; the configuration of each compound was assigned unequivocally. The diastereoisomeric couples of amino acids synthesized were tested, and their antiproliferative activity on MDR-positive and MDR-negative human cancer cell lines was evaluated.

Tamoxifen synergizes the antiproliferative effect of cisplatin in human ovarian cancer cells: enhancement of DNA platination as a possible mechanism.

We investigated the chemosensitizing activity of tamoxifen (TAM) on estrogen receptor negative ovarian cancer cell lines sensitive (A2780 WT) and resistant to cisplatin (CP) (A2780 CP3). Our results showed that the treatment of both cell lines with the association TAM + CP (concentration range 0.01-1 microN and 0.1-1 microgram/ml, respectively) results in a synergistic antiproliferative activity and a complete reversal of the acquired CP-resistant phenotype. We demonstrated that in A2780 cells the addition of TAM to CP treatment is able to significantly enhance at every tested CP dose (P < 0.001) the amount of platinum (Pt) bound to the DNA. Since Pt-DNA levels in the genome are clearly related to the growth inhibitory effect of CP (correlation value = 0.97, P < 0.001) in our experimental model, we hypothesized that TAM could act synergistically with CP and overcome the acquired CP-resistance by enhancing Pt binding to the DNA. We suggest that, from a clinical point of view, TAM may be usefully included in CP-based chemotherapy regimens for ovarian cancer patients since plasma concentrations of the drug capable of in vitro CP resistance modulation are achievable in vivo. A prospective clinical trial to verify the clinical usefulness of combined TAM + CP treatment in ovarian cancer patients refractory to prior Pt-based chemotherapy is now underway in our department.

Effects of dexamethasone on the growth and epidermal growth factor receptor expression of the OVCA 433 ovarian cancer cells.

We studied the correlation between dexamethasone (Dex) induced growth effects and modulation of epidermal growth factor receptor (EGFR) expression in OVCA 433 ovarian cancer cells. These cells express specific high and low affinity 125I-EGF binding sites and are growth stimulated by EGF. Dex exhibits mitoinhibitory effects by recruiting OVCA 433 cells in the G0-G1 phase of the cycle, but increases the number of both the high and the low affinity EGFR in a dose dependent manner. The maximal EGFR expression increase occurs after 24 h of Dex treatment consistently with Northern blot studies. The mitogenic activity of EGF in OVCA 433 cells is not affected by the presence of Dex. Moreover Dex growth inhibition occurs in JA1 cells, an ovarian cancer cell line which expresses unfunctional EGFR and which is unresponsive to EGF. Our results indicate that the Dex induced growth effects occur independently of EGFR expression.

The combination of quercetin and cytosine arabinoside synergistically inhibits leukemic cell growth.

It has been demonstrated that quercetin (3,3',4',5,7-pentahydroxyflavone) inhibits the growth of several cancer cell lines and that the antiproliferative activity of this substance is probably mediated through a binding interaction with type II estrogen binding sites (type II EBS). The effect of quercetin and cytosine arabinoside (Ara-C) alone or in combination, was tested on HL-60 cell growth. Quercetin significantly synergized the inhibitory activity of Ara-C on HL-60 cell growth while rutin, the 3-rhamnosylglucoside of quercetin, neither competed with [3H]estradiol for type II EBS nor was effective alone or in combination with Ara-C. Based on these results, we studied by a clonogenic assay the effect of quercetin and Ara-C alone and in combination on colony formation by human leukemic cells (CFU-L). In all cases both drugs exhibited a dose-related inhibition of CFU-L in a range of concentrations between 10 nM and 10 microM and 0.01 nM and 10 microM for quercetin and Ara-C, respectively. The combination of the two drugs resulted in a synergistic inhibitory activity on CFU-L. Considering that plasma concentrations of quercetin effective in vitro were obtained in vivo without any apparent side effects, we conclude that this report represents further experimental evidence that quercetin could be used in the treatment of acute leukemias.

Quercetin inhibits the growth of a multidrug-resistant estrogen-receptor-negative MCF-7 human breast-cancer cell line expressing type II estrogen-binding sites.

It has been demonstrated that the flavonoid quercetin (3,3',4',5,7-pentahydroxyflavone; Q) inhibits the growth of several cancer cell lines. There is evidence suggesting that the antiproliferative activity of this substance is mediated by the so-called type II estrogen-binding site (type II EBS). We looked for the presence of type II EBS and the effect of Q on the proliferation of an Adriamycin-resistant estrogen-receptor-negative human breast-cancer cell line (MCF-7 ADRr). By whole-cell assay using estradiol labelled with 6,7-tritium [( 3H]-E2) as a tracer, we demonstrated that MCF-7 ADRr cells contain type II EBSs. Competition analysis revealed that diethylstilbestrol (DES) and Q competed with similar potency for [3H]-Es binding to type II EBSs. The antiestrogen tamoxifen (TAM) competed for type II EBSs, albeit to a lesser extent than either DES or Q. Growth experiments demonstrated that Q and DES exerted a dose-dependent inhibition of cell proliferation in the range of concentrations between 10 nM and 10 microM, whereas TAM was less effective. Q could also inhibit colony formation in a clonogenic assay. Our results indicate that multidrug-resistant estrogen-receptor-negative MCF-7 cells express type II EBSs and are sensitive to the inhibitory effect of Q. This substance could be the parent compound of a novel class of anticancer agents.

Quercetin potentiates the effect of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: P-glycoprotein as a possible target.

This study demonstrates that the flavonoid quercetin (Q), a plant-derived compound with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of Adriamycin (ADR) on MCF-7 ADR-resistant human breast cancer cells. The effect of Q was dose-dependent at concentrations ranging between 1 and 10 microM. Since ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of Q and related flavonoids of Pgp activity in cytofluorographic efflux experiments with the fluorescent dye rhodamine 123 (Rh 123). Our results indicate that Q and 3-OMe Q (3',4',7-trimethoxyquercetin) but not the 3-rhamnosylglucoside of Q (rutin) inhibit the Pgp pump-efflux activity in a dose-related manner. Moreover, 10 microM Q reduces the expression of the immunoreactive Pgp in MCF-7 ADR-resistant cells as evaluated by cytofluorimetric assay. In conclusion, these findings provide a further biological basis for the potential therapeutic application of Q as an anti-cancer drug either alone or in combination with ADR in multidrug-resistant breast tumor cells.

In vitro evaluation of newly developed chalcone analogues in human cancer cells.

PURPOSE: Among flavonoids, chalcones have been identified as interesting compounds having chemopreventive and antitumor properties. We studied a panel of newly developed chalcone analogues (S1-S10) using MDA-MB 231 and MCF-7 ADRr breast cancer cells and the T-leukemic Jurkat cell line. Quercetin was used as the reference compound. METHODS: Antiproliferative activity was evaluated by cell counts performed after 72 h of exposure to the drugs. DNA analysis and redox activity were evaluated using flow cytometry. Apoptosis was assessed by morphological analysis, using YOYO-1 as DNA dye; p-glycoprotein function was ascertained by quantitating the efflux of rhodamine 123. RESULTS: All cells were sensitive to chalcone analogues yielding IC50 in micromolar concentrations with the following order regardless of the multidrug resistance (MDR) status: S1 > S2 > quercetin. S1 and S2, the most active compounds, were selected to evaluate their effect on the cell cycle, apoptosis, redox activity, and modulation of the p-glycoprotein function. No significant perturbation in cell cycle was seen with concentration up to 1 microM after 24 h. After 72 h a slight increase in G2/M block and DNA fragmentation occurred at 10 microM. Morphological analysis of apoptosis showed that chalcone analogues induced apoptosis to a higher extent than quercetin. Redox analysis demonstrated that all substances were able to increase intracellular thiol levels, which returned to baseline value after 24 h for all drugs except quercetin. Production of reactive oxygen species was essentially unaffected by all compounds. Finally, in MDR-positive MCF-7 ADRr cells chalcone analogues were unable to modulate p-glycoprotein function while quercetin was able to. CONCLUSIONS: Newly developed S1 and S2 chalcones have a different but higher antitumor activity than quercetin and could be considered as potential new anticancer drugs.

Antagonistic effect of the combination gemcitabine/topotecan in ovarian cancer cells.

The in vitro interaction between the new antimetabolite gemcitabine (GEM) and topotecan (TPT) was analyzed in A2780 ovarian cancer cells. The growth inhibitory effect was assessed after 3 days of drug exposure. GEM and TPT obtained in vitro IC50 values of 2.1 +/- 0.9 and 33.7 +/- 10.2 nM, respectively. The interaction between GEM and TPT was evaluated by exposing cancer cells at increasing doses of GEM (0.1, 1, and 10 nM) and TPT (1, 10, 100, and 1000 nM). Analysis of data about the interaction between GEM and TPT was performed by applying the isobole method. An antagonistic effect was noticed when GEM was combined with TPT in the tested concentration range. DNA analysis was also performed and showed an augmentation of cells blocked in the G2/M phase during TPT exposure, while an increase of blocked cells in the G0/1, phase was observed after GEM treatment. This latter effect was predominant when the two drugs were used in combination. We also investigated the effect of sequential exposure to drugs, pretreating A2780 cells for 24 h with TPT and then for 48 h with GEM, and, conversely, pretreating A2780 cells with GEM for 24 h and thereafter with TPT for 48 h. Both these combined sequential treatments showed an antagonist effect of the drugs' combination. Long-term growth inhibition effect was established by clonogenic assay performed after 10 days of culture after drug treatment. Also these data confirmed the antagonistic effect between GEM and TPT in A2780 ovarian cancer cells.

Biological evaluation on different human cancer cell lines of novel colchicine analogs.

Three new 7-0-substituted deacetamidothiocolchicine derivatives have been evaluated for their antitumor activity against various human tumor cell lines, some of which express the multidrug resistance (MDR) phenotype, for their impact on the cell cycle and their binding to tubulin. Colchicine and thiocolchicine were used as reference compounds. Thiocolchicine was the most active agent on MDR-negative cells in terms of growth inhibition, whereas for multidrug-resistant cells, thiocolchicone was the most active compound (IC50 = 14 nM). As indicated by statistical analysis, a perfect agreement for the potency order (IC50 values) of the compounds between all the MDR-negative cancer cells (k = 1.00), a poor agreement between MDR-positive and MDR-negative cancer lines, and a moderate agreement (k = 0.50) between the two resistant cancer cells MCF-7 ADRr and CEM VBL were observed. To gain further insight into the mechanism of the antitumor activity of colchicinoids, the most active compounds, colchicone and thiocolchicone, were selected to evaluate their effect on cell cycle, apoptosis, and tubulin interaction. The highest recruitment activity into the G21/M phase of the cell cycle was detected in thiocolchicone-treated breast cancer cells. Interestingly, after 72 h of culture, when the cell cycle block subsided, a consistent amount of DNA fragmentation, a hallmark of apoptosis, was evident. Morphological analysis of MCF-7 ADRr cells confirmed this hypothesis and revealed that thiocolchicone was able to induce apoptosis in this MDR-bearing model. We also demonstrated, using flow cytometry, that thiocolchicone interacts with alpha- and beta-tubulin, thereby affecting the expression of both subunits.

[Therapy of bacterial vaginosis. Prospective clinical study ot the efficacy and tolerability of quaternary ammonium salts vs. clindamycin]. / La terapia delle vaginosi batteriche. Studio clinico prospettico sull'efficacia e tollerabilità dei sali quaternari d'ammonio versus clindamicina.

BACKGROUND: To evaluate the efficacy and safety of intravaginal quaternary ammonium antimicrobial compounds (SQA) versus clindamycin 2% intravaginal cream (CL) in the treatment of bacterial vaginosis (VB). MATERIALS AND METHODS: One hundred-thirty-three patients affected by VB were enrolled in the study from January 1995 to October 1997. Patients were classified according to Amsel's criteria and/or to the indications of the Scandinavian Society of Bacterial Vaginosis. Twenty-three patients were initially excluded from the study, and 110 patients were randomized in two groups, SQA versus CL. Patients were reevaluated after 3 weeks, 3 months and 6 months from the end of therapy. The safety of treatment was also investigated. RESULTS: Of 110 patients, 59 were treated with SQA and 51 with CL. One hundred (90.9%) patients completed the therapy and were subjected to the first control after 3 weeks from the end of therapy. A significant reduction of most of the symptoms and all signs of VB was observed in the group treated with SQA. Similarly, a significant reduction of most of the symptoms (vaginal and urinary in particular) and all signs of VB was observed in the group treated with CL. The percentage of response was 86.7% for SQA group and 87.2% for CL group. Moreover, after 3 months from the end of therapy, 47.2% and 50% of the patients treated with SQA and CL, respectively, recurred, and after 6 months 78.5% and 75% of the patients recurred, respectively. CONCLUSIONS: SQA treatment conferred 86.7% of response after 3 weeks from the end of therapy, with poor side effects and a good compliance in good keeping with the results obtained with CL treatment.