Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow.

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family (HP1Hs alpha, HP1Hs beta and HP1Hs gamma), which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1Hs alpha, HP1Hs beta and HP1Hs gamma was determined by realtime RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow.

Apatite coating on anionic and native collagen films by an alternate soaking process.

The present study focuses on apatite coating on collagen films, with various different densities of carboxyl groups, using an alternate soaking process. Anionic collagen (AC), which has different densities of carboxylic groups compared to native collagen (NC), was obtained by hydrolysis of carboxyamides of asparagine and glutamine residues. From X-ray diffraction analysis, apatite was found to be coated on AC and NC films. Peaks ascribed to apatite were observed at 26 degrees and 32 degrees in the diffraction patterns of hydroxyapatite crystals. The amount of apatite coated on both AC and NC collagen films continued to increase up to 100 reaction cycles. However, there is a significant difference in apatite coating between the two films. The amount of apatite formed on the surface of AC film increased 1.24 times faster than on NC film. The scanning electron photomicrograph images of the mineralized NC and the AC film coatings formed after 100cycles show that regular porous apatite coating had formed within the collagen fibrils. These results suggest that the higher content of carboxyl groups in AC plays an effective role in the heterogeneous nucleation of apatite in the body environment.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer.

OBJECTIVES: Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. METHODS: Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 µg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. RESULTS: Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. CONCLUSIONS: Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

On the dielectric behaviour of collagen-algal sulfated polysaccharide blends: effect of glutaraldehyde crosslinking.

In this paper, impedance measurements in the frequency range from 10(-2) to 10(6) Hz are presented for collagen and algal sulfated polysaccharide crosslinked films. We are considering the development of new biomaterials which have potential applications in coating of cardiovascular prostheses, support for cellular growth and in systems for controlled drug delivery. The effect of crosslink sulfated polysaccharide on the physical chemical properties of collagen was studied using FT-infrared spectroscopy, differential scanning calorimetry (DSC), dielectric spectroscopy. The resulting films crosslinked with glutaraldehyde (GA) in concentrations of 0.001% and 0.05% when analysed by DSC, showed that the GA treatment not only left the thermal stability of the collagen unaffected, but it also decreased the thermal transition energy. Dielectric spectroscopy shows that the effect of the crosslink on the blend film was associated to the decrease and stabilization of the dielectric permittivity at low frequencies and decreased its conductivity.

Sensitivity and specificity of BCG scar reading in Brazil.

In a cross sectional survey within a community trial of BCG efficacy evaluation in Brazil, trained teams inspected children's upper arms and obtained information on BCG vaccination from guardian letters and vaccination cards. Nurses re-examined the sub-sample of children blindly. High agreement was found between the two scar readings (Kappa = 0.839). High sensitivity and low specificity was observed when guardian or card information was the gold standard. Sensitivity remained high when guardian and card information agreed. When disagreement occurred, sensitivity remained high and specificity was very low. BCG scar is a good indicator of BCG vaccination.

Rates of adverse reactions to first and second doses of BCG vaccination: results of a large community trial in Brazilian schoolchildren.

OBJECTIVE: To evaluate the incidence of adverse reactions to first and second bacille Calmette-Guérin (BCG) vaccination in schoolchildren. SETTING AND DESIGN: Enhanced surveillance in a Brazilian trial. Suspected reactions were reported to a nurse who visited cases and completed a standard form. RESULTS: Among 71341 schoolchildren studied, 33 reactions were reported. Of these, 25 fulfilled the criteria, resulting in a rate of one per 2854 vaccinations, with no deaths or BCG-osis. Reactions to second doses were more common than to first BCG vaccinations, but this difference was not statistically significant. CONCLUSIONS: Adverse reactions to a second dose of BCG may be more frequent than reactions to a first dose, but they are still rare events.

Elastosis and steroid receptors in primary breast cancer.

The association between the presence of both estrogen (ER) and progesterone (PR) receptors, and the grade of elastosis was evaluated in 100 primary breast carcinomas, 37% of which were from menopausal patients. ER levels were higher in post- than in premenopausal women but only when both ER and PR were present. Progesterone receptor levels were independent of menstrual status but the lowest PR positivity in premenopausal patients was observed during the late luteal phase. The extent of elastosis of the 100 tumors was: Grade 0 (absent), 24%; Grade 1 (small or moderate), 49%, and Grade 2 (marked), 27%. The extent of elastosis was not related to the patient's menstrual status. A significant association was observed between marked elastosis and the presence or absence of both ER and PR. No relationship was observed between the ER + PR and ER-PR+ subclasses and the extent of elastosis. Our data suggest that marked elastosis results from the presence of hormone-dependent cells in human breast carcinomas.

Periareolar mammaplasty: double skin technique with application of polyglactine or mixed mesh.

This paper presents the author's experience of 7 years in 254 patients operated on using a personal mammaplasty technique with a periareolar approach. The technique is based on the principle that the skin alone does not prevent early ptosis. This paper proposes the repositioning of all breast connective structures, treating the glandular set separately from the cutaneous lining, which is doubled, applying a circular flap of dermis with the central pedicle to the areolar region. For this purpose, the following structures have been employed, which, once repositioned and together, will act as a support network: (1) anterior pectoral fascia, (2) Cooper's intramammary ligament, (3) periareolar dermal flap, employed as skin inner lining. (4) application of absorbable polyglactine 910 or mixed mesh (with polyester) as a sandwich between both layers of skin, and (5) external cutaneous lining that composes the assembly of double skin.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer / Modelo de enxerto tumoral ortotópico em camundongos para avaliar os efeitos do calcitriol em câncer de mama

Abstract Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. Objectives To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. Methods Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 μg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. Results Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. Conclusions Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Resumo Os efeitos antiproliferativos de calcitriol foram observados em xenotransplantes de linhagens celulares de câncer de mama, entretanto, não foram ainda investigados em enxertos tumorais, consistindo de implantes em animais de amostras de câncer de mama recém-coletadas. Objetivos Estabelecer modelo de enxerto tumoral, a partir de amostra de câncer de mama recém-coletada e diretamente implantada em camundongos nude, para estudar o efeito do calcitriol. Métodos Amostras de câncer de mama de 12 pacientes foram implantadas ortotopicamente em camundongos nude. Os animais foram tratados com injeção intratumoral semanal de calcitriol 3 μg/Kg, a qual foi previamente associada com indução de pico sérico de calcitriol dentro do intervalo de nível terapêutico. Resultados A taxa de sucesso de pega do enxerto foi de 25%. Os enxertos tumorais foram estabelecidos de tumores agressivos com alta taxa de proliferação (HER2 positivo ou grau histológico 3) e as características do tumor original foram preservadas. O calcitriol induziu fortemente a expressão do gene alvo, CYP24A1, indicando que a via genômica da vitamina D está ativa nos enxertos tumorais, entretanto, não se observou diferenças na expressão de marcadores de proliferação e apoptose (incorporação de BrdU, expressão de Ki67, CDKN1A, CDKN1B e BCL2) nestas amostras altamente proliferativas. Conclusões Os enxertos tumorais parecem ser um modelo promissor para explorar outros esquemas e doses de calcitriol, considerando a heterogeneidade da doença e interações com o microambiente.

Polyanionic collagen membranes for guided tissue regeneration: Effect of progressive glutaraldehyde cross-linking on biocompatibility and degradation.

The ultimate goal of periodontal therapy is to control periodontal tissue inflammation and to produce predictable regeneration of that part of the periodontium which has been lost as a result of periodontal disease. In guided tissue regeneration membranes function as mechanical barriers, excluding the epithelium and gingival corium from the root surface and allowing regeneration by periodontal ligament cells. This report aims to study the effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen (PAC) membranes by conducting a histological evaluation of the tissue response (biocompatibility) and by assessing the biodegradation of subcutaneous membrane implants in rats. We studied six different samples: a PAC, a PAC mineralized by alternate soaking processes for either 25 or 75 cycles (PAC 25 and PAC 75, respectively) and these films cross-linked by GA. Inflammatory infiltrate, cytokine dosage, fibrosis capsule thickness, metalloproteinase immunohistochemistry and membrane biodegradation after 1, 7, 15 and 30 days were measured. The inflammatory response was found to be more intense in membranes without cross-linking, while the fibrosis capsules became thicker in cross-linked membranes after 30 days. The membranes without cross-linking suffered intense biodegradation, while the membranes with cross-linking remained intact after 30 days. The cross-linking with GA reduced the inflammatory response and prevented degradation of the membranes over the entire course of the observation period. These membranes are thus an attractive option when the production of new bone depends on the prolonged presence of a mechanical barrier.

Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancer patients.

In breast cancer patients submitted to neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, AC), expression of groups of three genes (gene trio signatures) could distinguish responsive from non-responsive tumors, as demonstrated by cDNA microarray profiling in a previous study by our group. In the current study, we determined if the expression of the same genes would retain the predictive strength, when analyzed by a more accessible technique (real-time RT-PCR). We evaluated 28 samples already analyzed by cDNA microarray, as a technical validation procedure, and 14 tumors, as an independent biological validation set. All patients received neoadjuvant chemotherapy (4 AC). Among five trio combinations previously identified, defined by nine genes individually investigated (BZRP, CLPTM1, MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2, and XLHSRF-1), the most accurate were established by RPL37A, XLHSRF-1 based trios, with NOTCH1 or NUP210. Both trios correctly separated 86% of tumors (87% sensitivity and 80% specificity for predicting response), according to their response to chemotherapy (82% in a leave-one-out cross-validation method). Using the pre-established features obtained by linear discriminant analysis, 71% samples from the biological validation set were also correctly classified by both trios (72% sensitivity; 66% specificity). Furthermore, we explored other gene combinations to achieve a higher accuracy in the technical validation group (as a training set). A new trio, MTSS1, RPL37 and SMYD2, correctly classified 93% of samples from the technical validation group (95% sensitivity and 80% specificity; 86% accuracy by the cross-validation method) and 79% from the biological validation group (72% sensitivity and 100% specificity). Therefore, the combined expression of MTSS1, RPL37 and SMYD2, as evaluated by real-time RT-PCR, is a potential candidate to predict response to neoadjuvant doxorubicin and cyclophosphamide in breast cancer patients.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas.

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancerpatients

In breast cancer patients submitted to neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, AC), expression of groups of three genes (gene trio signatures) could distinguish responsive from non-responsive tumors, as demonstrated by cDNA microarray profiling in a previous study by our group. In the current study, we determined if the expression of the same genes would retain the predictive strength, when analyzed by a more accessible technique (real-time RT-PCR). We evaluated 28 samples already analyzed by cDNA microarray, as a technical validation procedure, and 14 tumors, as an independent biological validation set. All patients received neoadjuvant chemotherapy (4 AC). Among five trio combinations previously identified, defined by nine genes individually investigated (BZRP, CLPTM1,MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2, and XLHSRF-1), the most accurate were established by RPL37A, XLHSRF-1based trios, with NOTCH1 or NUP210. Both trios correctly separated 86 percent of tumors (87 percent sensitivity and 80 percent specificity for predicting response), according to their response to chemotherapy (82 percent in a leave-one-out cross-validation method). Using the pre-established features obtained by linear discriminant analysis, 71 percent samples from the biological validation set were also correctly classified by both trios (72 percent sensitivity; 66 percent specificity). Furthermore, we explored other gene combinations to achieve a higher accuracy in the technical validation group (as a training set). A new trio, MTSS1, RPL37 and SMYD2, correctly classified 93 percent of samples from the technical validation group (95 percent sensitivity and 80 percent specificity; 86 percent accuracy by the cross-validation method) and 79 percent from the biological validation group (72 percent sensitivity and 100 percent specificity). Therefore, the combined expression of MTSS1, RPL37 and SMYD2, as evaluated by real-time RT-PCR, is a potential candidate to predict response to neoadjuvant doxorubicin and cyclophosphamide in breast cancer patients.

Carcinoembryonic antigen. A possible predictor of recurrence in cystosarcoma phyllodes.

Tissue carcinoembryonic antigen (CEA) and cytosolic estrogen and progesterone receptors were studied in 15 patients with cystosarcoma phyllodes (CSP) aiming at predicting recurrence of the tumor. Polyclonal (rabbit, monospecific) and monoclonal (mouse) antibodies anti-CEA were applied to formalin-fixed, paraffin-embedded tissue sections using an indirect (PAP) immunoperoxidase method. Estrogen receptors (ER) and progesterone (PR) receptors were determined by a charcoal-dextran method. ER was detected in 4 of 15 primary CSP (mean level, 22 fmol/mg protein). CEA was demonstrated exclusively in the epithelial components of 12 of 15 tumors. Strong expression of CEA was verified in eight tumors, six of which recurred locally one or more times. None of the seven tumors negative or weakly reactive for CEA had recurrences. No correlation was found between expression of tissue CEA and steroid receptor status of the tumors. Our data indicate that strong CEA expression in CSP correlates with tumor recurrence.

Steroid receptors in a group of Brazilian breast cancer patients.

Two hundred and forty-two primary breast cancers were assayed for estrogen receptors (ER). Of these, 202 were analyzed for progesterone receptors (PR) and 155 for glucocorticoid receptors (GR). ER was positive in 58% of the specimens; PR and GR were positive in 57%. A positive association was found between ER but not PR or GR frequency and age. Frequency of ER, PR, and GR positivity was approximately the same in premenopausal and postmenopausal women but ER content was much higher in postmenopausal women. About 70% of ER+ patients were also PR+ and GR+. Both frequency of PR positivity as well as average concentration, and frequency of GR positivity as well as average concentration were positively correlated with ER.

Steroid receptors in breast angiosarcoma.

The presence of receptors for estrogen, glucocorticoid and progesterone was determined in the cytosol of two breast angiosarcomas. Estrogen and glucocorticoid receptors were present in both of them. Progesterone receptors were present in one of the two tumors assayed. Occupied nuclear estrogen receptors have been found in the nuclear extracts of both tumors. Unoccupied nuclear receptors were found only in the progesterone-positive tumor. Density gradient analysis suggested that glucocorticoid and estrogen bindings were located predominantly in the 6S and 7 to 8S regions, whereas receptor for progesterone sedimented at 4S.

Anionic collagen: polymer composites with improved dielectric and rheological properties.

This work describes the preparation and characterization of anionic collagen composites with rhamsan and vinylidene fluoride-trifluorethylene with improved rheological and dielectric properties without loss of collagen secondary structure with an interaction occurring between both macromolecules of the composites. On a comparative basis, the force needed for the extrusion of anionic collagen:rhamsan composites was in the range from 0.088 to 0.080 J compared to that for collagen of 0.189 J. Anionic collagen:vinylidene fluoride-trifluorethylene composites were characterized, in the case of the 1:1 composite, by a pyroelectric coefficient of 1.89 x 10(-4) cm(-2) K(-1), which was significantly higher than those determined under the same conditions for native anionic collagen and vinylidene fluoride-trifluorethylene.

Steroid receptors in cystosarcoma phyllodes.

Seven cases of cystosarcoma phyllodes were studied. All but one of the patients were premenopausal. Histologic appearance permitted us to classify 5 as low-grade and 2 as high-grade tumors; 40% of the low-grade tumors were estrogen-receptor-positive (ER-positive), 100% were progesterone receptor-positive (PR-positive), and 60% glucocorticoid receptor-positive (GR-positive). In these tumors, mean PR values were 26.4 fmoles/mg protein; mean GR values were 27.2 fmoles/mg protein; mean ER values were 10,0 fmoles/mg protein. ER and PR were present in one of the high-grade tumors at slightly higher values than in low-grade tumors. GR was present in both high-grade tumors at a mean concentration of 275 fmoles/mg protein. Sucrose gradient analyses showed only the presence of 4S-specific receptors for ER and PR. GR receptors sedimented faster (6-7S).

Practical approaches to screening for cervical cancer.

The Program of the Fundação "Centro de Pesquisa de Oncologia" (FCPO) and the Instituto Brasileiro de Controle do Câncer (IBCC) in São Paulo has examined 980,977 women from 1970 to September 1985. Dysplasias, in situ, and invasive carcinomas were detected in 15,123 women (1.5%). The rates of false-negative and false-positive cytologic diagnoses of the program are 2.3% and 27%, respectively. The following epidemiological variables were evaluated: age, parity, age at first sexual intercourse, age at first pregnancy, education, and age at menarche. It was found that women at higher risk for cervical carcinoma in this population were those 26 years of age or older, with 4 or more pregnancies, women who had the first coitus before 18 years of age, and the first complete pregnancy before 19, as well as illiterate women or those who did not complete the first 4 years of primary school. In Brazil, the frequencies of cervical carcinomas are 25.8% for in situ and 72.2% for invasive (0.4 to 1). In this program, the corresponding percentages are 61.0% for in situ and 39.0% for invasive carcinomas (1.6 to 1). Including dysplasias, the percentages for Brazil are 56.3% for severe dysplasias plus in situ carcinomas and 43.7% for invasive carcinomas (1.3 to 1). In the FCPO/IBCC Program these percentages are 83.2% and 16.8% (5 to 1).

The use of 99mTc-phytate for sentinel node mapping in melanoma, breast cancer and vulvar cancer: a study of 100 cases.

Sentinel node mapping reduces surgical morbidity and allows the use of more accurate tumour staging techniques. Radionuclide studies are preferentially performed using small colloids, which have limited availability in our country. The possibility of using phytate for sentinel node mapping was raised because of the similarity between its biodistribution and that of nanocolloids in the reticulo-endothelial system. In this paper we evaluated the use of 99mTc-phytate for sentinel node mapping, correlating the histopathological results with the status of the rest of the lymph node chain in different malignant tumours. A total of 100 patients were studied. group 1 consisted of 62 patients with breast cancer, group 2 of 20 patients with melanoma and group 3 of 18 patients with vulvar carcinoma. Lymph node scintigraphy was carried out after injecting 99mTc-phytate subdermally, and the sentinel node projection was marked on the skin. After 18-24 h, intraoperative sentinel node localisation was performed using a gamma probe (combined with visual localisation using patent blue dye) in 75 patients, and lymph node dissection was then carried out. Radionuclide scintigraphy identified the sentinel node in 98% of all studies. Intraoperative detection using the gamma probe was equally efficient: group 1=93% (38/41), group 2=95% (18/19) and group 3=100% (15/15). The sentinel node was involved in 41%, 31% and 20% of cases in groups 1, 2 and 3, respectively. Among the patients with positive nodes, the sentinel node was the only one affected in 53% of group 1, 50% of group 2 and 67% of group 3 cases. The method's negative predictive value was 91% in group 1 and 100% in the other groups. One false-negative study occurred in a patient who had a multifocal tumour and an intraparenchymatous lymph node; another occurred in a patient with a macroscopically affected node found during surgery. There were no side-effects related to the 99mTc-phytate. It is concluded that scintigraphic and intraoperative sentinel node identification was satisfactorily performed using 99mTc-phytate. The results were comparable to those previously described in the literature using other radiopharmaceuticals. Easy availability and low cost justify the use of phytate in our practice.