Comparative analysis of in vitro proliferative, migratory and pro-angiogenic potentials of bovine fetal mesenchymal stem cells derived from bone marrow and adipose tissue.

Mesenchymal stem cells (MSCs) are found in virtually all tissues, where they self-renew and differentiate into multiple cell types. Cumulative data indicate that MSCs secrete paracrine factors that may play key roles in the treatment of various acute and chronic pathological conditions in diverse animal species including cattle. The aim of the present study was to compare the potentials for proliferation, migration and pro-angiogenesis of bovine fetal BM-MSCs and AT-MSCs under in vitro conditions. Growth curves and population doubling time (PDT) were determined for BM-MSCs and AT-MSCs in order to compare in vitro cell proliferation potentials. The ability of BM-MSCs and AT-MSCs to migrate was evaluated by scratch plate and transwell migration assays. The pro-angiogenic potential of conditioned medium from BM-MSCs and AT-MSCs was compared using an endothelial cell (EC) tubule formation assay. BM-MSCs displayed higher proliferation curves and doubled their populations in fewer days compared to AT-MSCs. No significant differences were detected in the number of migrant cells between BM-MSCs and AT-MSCs; however, a higher migration value was detected for BM-MSCs compared to fibroblasts (FBs), and a higher number of migrant cells were attracted by DMEM supplemented with 5% fetal bovine serum (FBS) compared to stromal cell-derived factor-1 (SDF-1). More tubules of ECs were formed after exposure to concentrated conditioned medium from AT-MSCs compared to BM-MSCs, FBs or DMEM controls. Despite common mesodermal origin, BM-MSCs display higher proliferative capacity and lower pro-angiogenic potential compared to AT-MSCs; however, both cell types possess similar migratory ability.

Is the snail shell repair process really influenced by eggshell membrane as a template of foreign scaffold?

Biominerals are inorganic-organic hybrid composites formed via self-assembled bottom up processes under mild conditions. Biominerals show interesting physical properties, controlled hierarchical structures and robust remodeling or repair mechanisms. Biological processes associated with biominerals remain to be developed into practical engineering processes. Therefore, the formation of biominerals is inspiring for the design of materials, especially those fabricated at ambient temperatures. The study described herein involves the influence of chicken outer eggshell membrane on the type of calcium carbonate (CaCO3) polymorph deposited on the shell of the land snail Helix aspersa during the repair process after an injury. A piece of snail shell was removed by perforating a hole from the largest body whorl. The operated area was left either uncovered or covered with either a thermoplastic flexible polyolefin-based film Parafilm® or a piece of chicken eggshell membrane. The repaired shells of control and experimental animals were analyzed using SEM, EDS, Raman and FTIR spectroscopies. We found that in the presence of eggshell membrane, the polymorph deposited on the substratum during the first hours resembles calcite, the polymorph present in eggshell normal formation, but at 24 and 48h, when snail mantle cells produced their normal organic matrix (mainly ß-chitin plus proteins and proteoglycans), the polymorph deposited is aragonite, the characteristic polymorph of Helix shell. Therefore, the eggshell membrane influences the type of polymorph, but only in the initial stages of biomineral deposition, before an organic matrix layer is deposited by the snail.

Austromegabalanus psittacus barnacle shell structure and proteoglycan localization and functionality.

Comparative analyzes of biomineralization models have being crucial for the understanding of the functional properties of biominerals and the elucidation of the processes through which biomacromolecules control the synthesis and structural organization of inorganic mineral-based biomaterials. Among calcium carbonate-containing bioceramics, egg, mollusk and echinoderm shells, and crustacean carapaces, have being fairly well characterized. However, Thoraceca barnacles, although being crustacea, showing molting cycle, build a quite stable and heavily mineralized shell that completely surround the animal, which is for life firmly cemented to the substratum. This makes barnacles an interesting model for studying processes of biomineralization. Here we studied the main microstructural and ultrastructural features of Austromegabalanus psittacus barnacle shell, characterize the occurrence of specific proteoglycans (keratan-, dermatan- and chondroitin-6-sulfate proteoglycans) in different soluble and insoluble organic fractions extracted from the shell, and tested them for their ability to crystallize calcium carbonate in vitro. Our results indicate that, in the barnacle model, proteoglycans are good candidates for the modification of the calcite crystal morphology, although the cooperative effect of some additional proteins in the shell could not be excluded.

Epoxy putty external skeletal fixation in a tibiotarsal fracture of a wild choroy parakeet (Enicognathus leptorhynchus) / Fixação externa com massa epóxi em uma fratura tibiotársica de um periquito choroy selvage (Enicognathus leptorhynchus)

Tibiotarsal fractures are common in birds because in most birds this is the longest, most exposed bone in the leg. Transverse fractures are most common and rotational and shear forces must be stabilized in order to achieve good bone regeneration. A 230g male Slender-billed Parakeet or Choroy parakeet (Enicognathus leptorhynchus), with more than five years of age, was received with non-weight bearing lameness with 24 hours duration. X-rays were taken, and these revealed a closed, complete, non-comminuted transverse fracture of the distal diaphysis of the left tibiotarsal bone. Fixation was planned with 10-minute fast-setting epoxy putty. In order to assess the temperature of polymerization of the epoxy dough and the possibility of heat-bone necrosis, the temperature was recorded every 30 seconds for 12 minutes with three different amounts of the epoxy material in an ex vivo test. The temperature of the pieces reached a peak of 50-60ºC, where the highest peak corresponds to the highest amount of material. When approximately 6g of putty were used, the peak temperature reached only 51ºC. This peak changed to 58ºC when 4 times more epoxy was mixed and measured. If the temperature of the pins inserted in the bone exceeds 70ºC, bone necrosis could occur. In light of these results, the fracture was treated with 6 g of epoxy putty that was allowed to polymerize over a 1A 2/2 external skeletal fixation, with 1-mm pins bent at 90º and joined together with cerclage wire. At 6 weeks after surgery the bird had formed a good primary bone callus, and the external fixators were therefore removed. With this approach a satisfactory recovery of the patient was achieved with normal use of the affected limb.

Fraturas tibiotársicas são comuns em aves, pois, na maioria das aves, o tibiotarso é o osso mais longo e exposto nesses animais. Fraturas transversais são mais comuns, sendo que as forças de rotação e cisalhamento devem ser estabilizadas de modo a alcançar uma boa regeneração óssea. Um periquito macho do tipo psitacídeo de 230g, com mais de 5 anos, foi recebido apresentando claudicação a 24 horas não devido a transferência de carga. Os exames radiográficos revelaram uma fratura fechada completa, não triturada, transversal de diáfise distal do osso esquerdo tibiotársico. A fixação foi feita com massa epóxi de endurecimento rápido (10 minutos). A fim de avaliar a temperatura de polimerização da massa epóxi e a possibilidade de necrose desses ossos, a temperatura foi registrada a cada 30 segundos durante 12 minutos, com três quantidades diferentes de epóxi em teste in vivo. A temperatura das amostras fragmentadas atingiu um pico de 50-60ºC, em que o pico mais elevado corresponde à maior quantidade de massa epóxi. Quando foram utilizados cerca de 6g de massa de vidraceiro, o pico de temperatura foi de apenas 51ºC. Esse pico foi alterado para 58ºC quando se aumentou a quantidade de massa epóxi quatro vezes. Se a temperatura dos pinos inseridos no osso for superior a 70ºC, pode ocorrer necrose do osso. À luz desses resultados, a fratura foi tratada com 6g de massa epóxi deixada polimerizar sobre uma fixação externa, em configurações 1A 2/2 usando pinos de 1mm, dobrados em 90º e unidos com fios de cerclage. Após 6 semanas da cirurgia, o pássaro tinha formado um calo ósseo primário, sendo os fixadores externos removidos. Com essa abordagem, conclui-se que houve a restauração das funções normais do membro e total recuperação da ave.

Only in patients with hormone-dependent breast infiltrating ductal carcinomas, CA15.3 serum levels are inversely correlated with the immunohistochemical expression of Bcl2.

OBJECTIVE: To study possible association between serum CA15.3 levels and immunohistochemical expression of Bcl2 in women affected by infiltrating ductal breast carcinomas. MATERIALS AND METHODS: Two hundred and fifty consecutives women with breast infiltrating ductal carcinomas, aged between 37 and 83 years were included in this study. Serum CA15.3 was determined by electro-chemoluminescence assay (ECLIA-Elecsys 170 Roche). Immunohistochemical staining on tissue sections of 4-5 microns was done by the EnVision method with a heat-induced antigen retrieval step. Antibody used for Bcl2 was (124, Dako, dilution 1/150). Bcl2 expression was assessed as negative (-), weak positive (+) or strong positive (++). RESULTS: In the study group, serum CA15.3 concentrations ranged between 1 and 1743 U/ml, with 25, 50 and 75 percentiles of 12.7, 17.6 and 24.3 U/ml respectively. Serum CA15.3 concentrations were higher in Bcl2 negative cases than in Bcl2 + and Bcl2 ++. We found statistically significant differences between subgroups Bcl2 negative and Bcl2 ++ (p=0.044), between Bcl2 + Bcl2 ++ (p=0.039) and between Bcl2 ++ and Bcl2 -/+ (p=0.013). When we considered 25 U/mL as the threshold of positivity, antigen values>25 U/ml were more frequent in tumors Bcl2- than in Bcl2 ++ (20/52 vs 29/170, p=0.001). The same behavior was observed when comparing the subgroups -/+ with ++ (p=0.001). A very important aspect of our work was that this CA15.3 behavior in relation to the immunohistochemical expression of Bcl2 was maintained in hormone-dependent tumors (ER+), but not in hormone-independent ones. CONCLUSIONS: The results led us to the following consideration: In women affected by infiltrating ductal breast carcinomas, serum levels of CA15.3 associated inversely, both qualitatively and quantitatively, with the immunohistochemical expression of Bcl2, but this fact exists only in hormone-dependent tumors.

Breast carcinomas with hyperprolactinemia at the time of diagnosis-clinico-biological association.

AIM: To analyse association between preoperative hyperprolactinemia serum levels and clinical and biological features of breast tumors. METHODS: Serum levels of prolactin were measured in 253 women with invasive breast cancer. Clinical and biological parameters analysed were age, size, lymph node involvement, distant metastasis and immunohistochemical expression of estrogen receptor, progesterone receptor, androgen receptor, bcl-2, p53 and Ki67. RESULTS: In ductal carcinomas hyperprolactinemia were associated with high age (p = 0.017), and with bcl-2 + + + expression (p = 0.017). Furthermore, serum prolactin values were significantly higher in bcl-2 +++ cases vs negative (p = 0.029); the same happened when we considered the positivity threshold of 25 ng/mL (p = 0.015). CONCLUSION: Is possible to detect in 6% of infiltrating ductal breast carcinomas hyperprolactinemia (>25 ng/mL), being associated only with increasing age, but not with other clinical or biological factors; and 2) the most surprising data was the association between prolactinemia (qualitative (>25 ng/mL) and quantitative) and intense bcl-2 tissue expression, which suggests that, probably, this (prolactinemia) is not a sign of worse prognosis and evolution.

Evaluation of a multilayered chitosan-hydroxy-apatite porous composite enriched with fibronectin or an in vitro-generated bone-like extracellular matrix on proliferation and diferentiation of osteoblasts.

The use of extracellular matrix (ECM) molecules from tissues is an interesting way to induce specific responses of cells grown onto composite scaffolds to promote adhesion, proliferation and differentiation. There have been several studies on the effects on cell proliferation and differentiation of osteoprogenitor cells cultured onto composites, either adding some ECM molecules or grown in the presence of growth factors. Other studies involve the use of osteoblasts cultured on a three-dimensional (3D) matrix, enriched with ECM molecules produced by the same cells grown previously inside the composite. Here, the effect of enrichment of a novel multilayered chitosan-hydroxyapatite composite with ECM molecules produced by osteoblasts, or the addition of 25 or 50 µg/ml fibronectin to the composite, on proliferation and differentiation of osteoblasts cultured on these composites was studied. The results showed an increase in the number of osteoblasts from day 1 of culture, which was higher in the group grown onto composites enriched with the highest concentration of fibronectin or with ECM molecules produced naturally by osteoblasts cultured previously on them, when compared with the control group. However, this increment tended to decline in all groups after day 7 of culture, the day when they reached the highest peak of proliferation. Differentiation expressed as alkaline phosphatase activity followed the proliferation pattern of the cells cultivated on the scaffolds. The results demonstrate the potential offered by these enriched 3D multilayered composites for improving their ability as bone grafting material.

The Fanconi anemia family of genes and its correlation with breast cancer susceptibility and breast cancer features.

Fanconi anemia (FA) family of proteins participates in the DNA repair pathway by homologous recombination, and it is currently formed by 13 genes. Some of these proteins also confer susceptibility to hereditary breast and ovarian cancer (HBOC), since FANCD1 is the BRCA2 breast cancer susceptibility gene, and FANCN/PALB2 and FANCJ/BRIP1 explain 2% of non-BRCA1/2 HBOC families. Thus, there is an important connection between FA and BRCA pathways. In a previous case-control association study analysing FANCA, FANCD2 and FANCL, we reported an association between FANCD2 and sporadic breast cancer (BC) risk (OR = 1.35). In order to know whether variants in other FA genes could also be involved in this association, we have extended our study with the rest of FA genes and some others implicated in the BRCA pathway. We have also analyzed the correlation with survival, nodal metastasis and hormonal receptors (ER- and PR-). A total of 61 SNPs in ten FA genes (FANC-B, -C, -D1, -E, -F, -G, -I, -J, -M, -N) and five FA related genes (ATM, ATR, BRCA1, H2AX and USP1) were studied in a total of 547 consecutive and nonrelated sporadic BC cases and 552 unaffected controls from the Spanish population. Association analyses reported marginal statistically significant results with the minor allele of intronic SNPs in three genes: BRCA1, BRCA2/FANCD1, and ATM. Survival association with SNPs on FANCC and BRCA2/FANCD1 genes were also reported. Sub-group analyses revealed associations between SNPs on FANCI and ATM and nodal metastasis status and between FANCJ/BRIP1 and FANCN/PALB2 and PR- status.

Eggshell membrane as a biodegradable bone regeneration inhibitor.

The efficiency of chicken eggshell membranes combined with a minimally invasive small osteotomy procedure of the ulna to accomplish an efficient release of the radius so that it can continue to grow in an unstressed manner was tested in rabbits. Eggshell membranes were extracted from chicken eggs, rinsed, dried and sterilized with ethylene oxide for 24 h. For reactivity testing, four separate subcutaneous pockets were created in 10 rats in the paravertebral region by blunt dissection and eggshell membranes were implanted in two of them. After 1-16 weeks, the implants were retrieved with the surrounding soft tissues and submitted to histological examination. Subsequently, 10 rabbits were anaesthetized and a complete 0.5 mm wide osteotomy was performed in both the right and the left distal ulna. A piece of eggshell membranes was interposed in the osteotomy site of one ulna. The opposite osteotomized ulna was left as a negative control. The rabbits were injected with oxytetracycline at the time of surgery and this was repeated every 7 days for labelling new bone formation. After 1-16 weeks, ulnar osteotomized regions were histologically examined. After histological, fluorescence microscopy and radiological evaluation, we demonstrate here for the first time that eggshell membranes as interpositional material in rabbit osteotomized ulnar experiments acted as an active barrier against bone bridging. The degradation of the eggshell membrane, due to host reaction, appeared sufficiently late to cause the desirable delay of bone healing that is compatible with the time needed for a corrective response.

CA15.3 sérico y proliferación celular en carcinomas ductales infiltrantes de mama / Preoperative CA15.3 serum levels and cellular proliferation in patients having infiltrating ductal carcinomas of the breast

Objetivo. Estudiar la posible correlación entre las concentraciones séricas preoperatorios de CA15.3 y laproliferación celular medida por la fase de síntesis (FS) en carcinomas ductales infiltrantes de mama (CDI).Material y métodos. El grupo estudio incluyó 79 pacientesde edades comprendidas entre los 39 y 86 años (64,8 ± 11,8). La ploidía y FS fueron determinadas por citometría de flujo en muestras en fresco (Fascam. Beckton Dikinson. EE.UU.). Resultados. Tomando como dintel de positividad para la FS el valor de 7 %, que representa la mediana obtenida previamente en un grupo de 321 CDI (i: 0,8-51,2; 9,3 ± 7,9; percentiles 25 y 75; 4,3 y 11,8 %), observamos que las concentraciones del marcador fueron mayores (p: 0,015) en los casos con menor proliferación celular. Esto mismo se constató al valorar cualitativamente (> 30 U/ml) el marcador. Asimismo, las concentraciones de CA15.3 aumentaron significativamente (p = 0,007) al pasar la FS de < 4,3 % a FS comprendida entre 4,3 y 7,1 %, para luego descender (p = 0,010) en los casos con FS entre 7,11 y 11,8% y no modificarse cuando aquella fue > 11,8 %. Este mismo comportamiento lo observamos en los tumores sin afectación axilar. Conclusiones. a) La liberación del CA15.3 ocurre cuando la FS se incrementa hasta alcanzar el valor del 7,1 %, para luego ir disminuyendo aunque aquella aumente, y b) este mismo comportamiento del marcador con la fase S se constató cuantitativamente en los tumores aneuploides y sin afectación axilar

Objective. To study the possible correlations betweenthe preoperative CA15.3 serum levels and the cellularproliferation, measured by S-phase (SP), in patients having infiltrating ductal carcinomas (IDC) of the breastMaterial and methods. The study group included 79 patients with an age ranged between 39 and 86 yrs (64,8 ± 11,8). Ploidy and S-phase were measured by cytometry (Fascam. Beckton Dikinson. USA) in fresh samplesResults: Using as cut-off for SP the value of 7 %, which represents the median obtained previously in 321 patients with IDC (r: 0,8-51,2; 9,3 ± 7,9; percentiles 25 y 75; 4,3 y 11,8 %), we can observed that the antigenic levels were higher (p:0,015) in the tumors with low SP. These same behavior was noted when 30U/ml was used as cut-off for CA15.3. Likewise, the levels of the tumor marker increased significantly (p:0,007)when the SP moved from < 4,3 % to 7,1 %, to decrease later (p:0,010) when the SP value was comprised between 7,11% and 11,8 %. The same behavior of this tumor marker in relation to the SP was noted in tumors without axillary involvement tumors, as well as in aneuploid carcinomas. Conclusion: a) Release of CA15.3 happens when SP increases to rise the 7,1 % value, to decrease later although that goes on increasing, and b) The same behaviour of this marker with the S-phase was observed in tumors without axillary involvement, as well as in aneuploid carcinomas

[Preoperative CA15.3 serum levels and cellular proliferation in patients having infiltrating ductal carcinomas of the breast]. / CA15.3 sérico y proliferación celular en carcinomas ductales infiltrantes de mama.

OBJECTIVE: To study the possible correlations between the preoperative CA15.3 serum levels and the cellular proliferation, measured by S-phase (SP), in patients having infiltrating ductal carcinomas (IDC) of the breast MATERIAL AND METHODS: The study group included 79 patients with an age ranged between 39 and 86 yrs (64,8 +/- 11,8). Ploidy and S-phase were measured by cytometry (Fascam. Beckton Dikinson. USA) in fresh samples RESULTS: Using as cut-off for SP the value of 7 %, which represents the median obtained previously in 321 patients with IDC (r: 0,8-51,2; 9,3 +/- 7,9; percentiles 25 y 75; 4,3 y 11,8 %), we can observed that the antigenic levels were higher (p:0,015) in the tumors with low SP. These same behavior was noted when 30U/ml was used as cut-off for CA15.3. Likewise, the levels of the tumor marker increased significantly (p:0,007) when the SP moved from < 4,3 % to 7,1 %, to decrease later (p:0,010) when the SP value was comprised between 7,11 % and 11,8 %. The same behavior of this tumor marker in relation to the SP was noted in tumors without axillary involvement tumors, as well as in aneuploid carcinomas. CONCLUSION: a) Release of CA15.3 happens when SP increases to rise the 7,1 % value, to decrease later although that goes on increasing, and b) The same behaviour of this marker with the S-phase was observed in tumors without axillary involvement, as well as in aneuploid carcinomas.

The inflammatory response recapitulates phylogeny through trophic mechanisms to the injured tissue.

The post-traumatic local acute inflammatory response is described as a succession of three functional phases of possible trophic significance: 1. Nervous or immediate (ischemia-reperfusion); 2. Immune or intermediate (infiltration by inflammatory and bacterial cells) and 3. Endocrine or late (angiogenesis with regeneration and/or cicatrization). Each of these phases emphasizes the trophic role of the mechanisms in the damaged tissue. Hence, the nervous phase is predominated by nutrition by diffusion; in the immune phase trophism is mediated by inflammatory cells and bacteria and, finally, in the endocrine phase, the blood circulation and oxidative metabolism play the most significant nutritive role. Since these trophic mechanisms are of increasing complexity, progressing from anoxia to total specialization in the use of oxygen to obtain usable energy, it could be speculated that they represent the successive reappearance of the stages that take place during the evolution of life on Earth, from ancient times without oxygen. In this sense, the inflammatory response could recapitulate phylogeny through the successive expression of pathophysiologic mechanisms that have a trophic meaning to the injured tissue.

Aspectos biológicos del grado nuclear en los carcinomas mamarios / No disponible

Objetivo: estudiar cómo el grado nuclear (GN) influye enlas características clínico-biológicas de los carcinomas ductalesinfiltrantes de mama.Material y métodos: se incluyeron 236 pacientes, de loscuales 87 eran GN1, 93 GN2 y 56 GN3. Hemos analizado lasconcentraciones citosólicas de receptor de estrógenos (RE), deprogesterona (RP), pS2, catepsina D y ácido hialurónico, asícomo las de este y el receptor del factor de crecimiento epidérmicoen las membranas celulares. Hemos considerado,además, el tamaño, afectación axilar, metástasis a distancia,ploidía y fase de síntesis celular.Resultados: los tumores grado 3 mostraron, en relación alos 1, mayores concentraciones de catepsina D (p = 0,021) ymenores de receptor de estrógenos (p = 0,033), siendo, asimismo,más frecuentemente proliferativos (Fase S > 7%; p =0,001). Al considerar los carcinomas mamarios en función dela hormonodependencia, pudimos ver, en los tumores RE+,que los GN3 fueron más frecuentemente proliferativos (p =0,052), mientras que en los casos RE- cursaron con mayoresconcentraciones de catepsina D (p = 0,026). Similares hallazgosobtuvimos al tener presente la afectación ganglionar axilar.Conclusiones: a) el paso de GN 1 a 3 se asocia con menorhormonodependencia, mayor proliferación y superiores concentracionesde catepsina D; b) estas diferencias biológicas difierencuando los tumores se clasifican en función de la hormonodependenciay afectación ganglionar axilar: en los casosRE+ o N- la transición del GN se asocia preferentemente a laproliferación celular, mientras que en los casos RE- o N+ lohace con la catepsina D(AU)

Objectives: to study the association of nuclear grade (NG)with specific clinical and biological parameters which may influencethe clinical behaviour of infiltrating ductal carcinomasof the breast (IDC)Material and methods: we analyzed in 236 tissue samplesthe cytosolic concentrations of estrogen receptor (ER), progesteronereceptor (PR), pS2, cathepsin D and hyaluronic acid(HA), as well as those of the epidermal growth factor receptor(EGFR) and HA in the cell membrane fraction. Likewise, weconsidered size, ploidy, S-phase fraction and axillary node involvementand distant metastasis as variables of the study.Results: the transition from NG1 to NG3 was accompaniedby greater cellular S-Phase > 7% (p = 0.001), higher cathepsinD levels (p = 0.021) and lower ER concentrations (p =0.033). When we considered the positivity for ER (> 10fmol/mg prot.), we can observe that, in the IDC-ER+ tumors,NG3 was accompanied by greater SF+ (p = 0.002), whereasin the IDC-ER- cases it was associated with higher cathepsinDlevels (p = 0.026). Similar features were observed when axillarynodal involvemente was taken accountConclusions: these results led us to consider that NG is associatedwith some clinical-biological changes (greater proliferation,higher cathepsin D levels) that may help to explain itsvalue as a prognostic factor in breast carcinomas. Nevertheless,these biological associations depends on the hormonodependeceor axillary nodal invasion of the tumors. In ER-positiveor N-negative, NG3 was associated preferently with agreater cellular proliferation, whereas in ER-negative or N-positiveit was associated with higher cathepsin D levels(AU)

[Preoperative serum CA15.3 and CEA levels and clinical-biological parameters in breast tumors]. / Concentraciones séricas preoperatorias de antígeno carbohidrato 15.3 y antígeno carcinoembrionario y características clínico-biológicas tisulares de los carcinomas mamarios.

OBJECTIVE: To study the possible associations between preoperative serum CEA and CA15.3 levels and different clinical-biological parameters of infiltrating ductal carcinomas of the breast (IDC). PATIENTS AND METHOD: Preoperative serum CA15.3 and CEA levels were determined in 255 and 224 females, respectively, having IDC. We assayed the cytosolic levels of estrogen receptor, pS2, cathepsin D and hyaluronic acid, as well as the levels of epidermal growth factor receptor in cell surfaces. Tumor size, axillary involvement (N), distant metastasis (M), histological grade (HG) and cellular S-phase (SP) were taken into account. RESULTS: 22 IDC were positive for CA15.3 (> 40 U/ml) and they had greater global tumor size (p < 0.001), > 2 cm (p: 0.005) and distant metastasis (p < 0.001). 19 IDC were positive for CEA (> 4 ng/ml) and they had greater global tumor size (p < 0.001), > 2 cm (p < 0.001), > 5 cm (p: 0.052) and global S-phase values (p: 0.062), and were more frequently N + (p: 0.045), > 3N + (p: 0.001) and > 10N + (p < 0.001), M + (p: 0.004), HG3 (p: 0.091) and SP > 7 % (p: 0.006). CONCLUSIONS: Our results led us to the following: In patients having IDC of the breast, preoperative serum CA15.3 levels are associated with greater tumor size and distant metastasis, while pre-treatment CEA serum levels are associated moreover with axillary involvement; we have not observed any correlation between serum levels of both antigens and the hormonal status of the tumor; these results had physiopathological interest but reduced clinical value in pretreatment breast carcinomas due to their low sensitivity of both markers. However, they are useful in the follow-up of the patients when assessing serum concentrations of both markers, since they contribute to knowing the patients' clinical status better.

FANCD2 associated with sporadic breast cancer risk.

Several components of the Fanconi anaemia (FA) family of proteins allow the formation of the DNA repair complex foci formed by proteins such as BRCA1/2 and RAD51. Because the genes that participate in the DNA repair pathway have been described as low-penetrance breast cancer susceptibility genes, we postulated that variants in FA genes could also be associated with sporadic breast cancer risk. We studied seven SNPs in FANCA, FANCL and FANCD2 in a total of 897 consecutive and non-related sporadic breast cancer cases and 1033 unaffected controls from the Spanish population. We observed a statistically significant association with sporadic breast cancer for the variant rs2272125 (L1366L) located on FANCD2 (OR per allele=1.35; 95% C.I. 1.09-1.67; P=0.005). Both haplotype and diplotype analyses confirmed this association, where one haplotype and pooled diplotypes carrying it were associated with more than 4-fold risk (P=0.007 and P=0.006, respectively). Screening for potential causal variants in FANCD2 was performed, detecting one in the putative promoter region, which is located in a phylogenetically conserved motif with consensus binding sites for some transcriptional factors, suggesting a functional implication. Our data indicate that a relationship between FANCD2 and sporadic breast cancer risk may exist.

Concentraciones séricas preoperatorias de antígeno carbohidrato 15.3 y antígeno carcinoembrionario y características clínico-biológicas tisulares de los carcinomas mamarios / Preoperative serum CA15.3 and CEA levels and clinical-biological parameters in breast tumors

Objetivo. Estudiar las posibles correlaciones entre las concentraciones séricas pretratamiento del antígeno carcinoembrionario (CEA) y antígeno carbohidrato (CA) 15.3 y diferentes parámetros clínico-biológicos no titulares de carcinomas ductales infiltrantes de mama (CDI).Pacientes y método. El grupo de estudio incluyó 255 y 224 mujeres afectas de CDI pretratamiento en las que determinamos, mediante sendos IRMAS, las concentraciones séricas de CA 15.3 y CEA respectivamente. A nivel tisular dosificamos las concentraciones citosólicas de receptor de estrógenos, pS2, catepsina D y ácido hialurónico, así como las del receptor del factor de crecimiento epidérmico (EGFR) en las membranas celulares. Tuvimos también presente el tamaño, afectación ganglionar axilar (N), metástasis a distancia (M), grado histológico (GH) y fase de síntesis celular (FS).Resultados. Los 22 CDI con concentraciones de CA15.3 > 40 U/ml mostraron mayor tamaño global (p 2 cm (p: 0,005), así como metástasis a distancia (p 4 ng/ml) cursaron con mayor tamaño global (p 3 N + (p: 0,001) y > 10N + (p 2 cm (p 5 cm (p: 0,052), GH3 (p: 0,091) y FS > 7 % (p: 0,006).Conclusiones. Nuestros resultados inducen a las siguientes consideraciones: las concentraciones séricas pretratamiento del CA15.3 en mujeres afectas de CDI de mama se asocian con un mayor tamaño y metástasis a distancia, mientras que las de CEA lo hacen, además, con la afectación ganglionar; no hemos constatado una relación con el estatus hormonal del tumor y las concentraciones séricas de ambos marcadores tumorales; estos resultados son de interés fisiopatológico, de reducido valor clínico en los carcinomas mamarios pretratamiento, dada la baja sensibilidad de ambos marcadores, pero de utilidad en el seguimiento de los pacientes a la hora de valorar las concentraciones séricas de ambos marcadores, pues pueden contribuir a conocer de un modo más exacto su situación

Objective. To study the possible associations between preoperative serum CEA and CA15.3 levels and different clinical-biological parameters of infiltrating ductal carcinomas of the breast (IDC).Patients and method. Preoperative serum CA15.3 and CEA levels were determined in 255 and 224 females, respectively, having IDC. We assayed the cytosolic levels of estrogen receptor, pS2, cathepsin D and hyaluronic acid, as well as the levels of epidermal growth factor receptor in cell surfaces. Tumor size, axillary involvement (N), distant metastasis (M), histological grade (HG) and cellular S-phase (SP) were taken into account.Results. 22 IDC were positive for CA15.3 (> 40 U/ml) and they had greater global tumor size (p 2 cm (p: 0.005) and distant metastasis (p 4 ng/ml) and they had greater global tumor size (p 2 cm (p 5 cm (p: 0.052) and global S-phase values (p: 0.062), and were more frequently N + (p: 0.045), > 3N + (p: 0.001) and > 10 N + (p 7 % (p: 0.006).Conclusions. Our results led us to the following: In patients having IDC of the breast, preoperative serum CA15.3 levels are associated with greater tumor size and distant metastasis, while pre-treatment CEA serum levels are associated moreover with axillary involvement; we have not observed any correlation between serum levels of both antigens and the hormonal status of the tumor; these results had physiopathological interest but reduced clinical value in pretreatment breast carcinomas due to their low sensitivity of both markers. However, they are useful in the follow-up of the patients when assesing serum concentrations of both markers, since they contribute to knowing the patients'clinical satus better

[Expression of epidermal growth factor receptor (EGFR) in hormone-independent breast cancers]. / La expresión del receptor del factor de crecimiento epidérmico (EGFR) en carcinomas mamarios hormonoindependientes.

OBJECTIVE: To determine the expression of epidermal growth factor receptor (EGFR) in ER-negative and Pg-R negative infiltrating ductal carcinomas of the breast (IDC) and to analyze the possible relationship between the EGFR positivity and some clinico-biological parameters of tumors. MATERIAL AND METHODS: EGFR was measured by a single point radioligand assay in the cell surfaces of 115 ER-negative and Pg-R-negative (< 10 fmol/mg prot.) IDC. We measured also the cytosolic concentrations of pS2, cathepsin D, tissue-type plasminogen activator (t-PA) and hyaluronic acid (AH), as well as the levels of AH in cell surfaces. Tumor size, axillary involvement, distant metastasis, histological grading, ploidy and S-phase (SP) were taken account. RESULTS: Using as cut-off for EGFR a value of 5 fmol/mg prot., we can observed that IDCs -EGFR + had greater global values of S-phase (p: 0.005) and were more frequently metastastatic (p: 0.004), SP > 7 % (p < 0.001) and SP > 14 % (p: 0.077); likewise, they were lower frequently pS2-positive (p < 0.01) and t-PA-positive (p < 0.01). During the follow up time (median 85 months), the number of recurrences was higher in EGFR-positive than in EGFR- negative tumors (14/41 frente a 1/29; p: 0.002), but there was not differences in the number of deaths by the tumor. CONCLUSIONS: 1) The EGFR-positivity in ER-negative and PgR-negative IDCs is associated with distant metastasis, greater cellular proliferation (SP), lower positivity for pS2 and t-PA and greater number of recurrences. 2) Using 10 fmol/mg prot. as cut-off, we observed the same findings, except the change in cellular proliferation. 3) Our findings support the possible use of EGFR as a prognostic parameter in those breast carcinomas.

Estrogen and progesterone receptor gene polymorphisms and sporadic breast cancer risk: a Spanish case-control study.

Estrogens, and to a lesser extent progesterones, influence the proliferation, differentiation and physiology of breast tissue as well as the development and progression of breast cancer. Genetic variants in the steroid hormone receptor genes ESR1 and PGR (belonging to the nuclear receptor superfamily) could therefore modify sporadic breast cancer susceptibility. Two studies have shown a protective effect associated with variants in ESR1 in 2 distinct populations. We studied 4 single nucleotide polymorphisms (SNPs) in ESR1 and 4 in PGR in 550 consecutive and unrelated sporadic Spanish breast cancer patients and 564 healthy Spanish controls. We observed a dominant protective effect for the S10S variant in ESR1, with an estimated odds ratio (OR) of 0.75 (95% CI = 0.58-0.97; p = 0.03) although functional studies did not show changes in the RNA stability. A small subset of individuals carried a haplotype combination that corroborates this protection. No other SNP considered in either gene was found to be associated with sporadic breast cancer. Our results obtained in a European population confirm the protective role of the S10S variant in ESR1, previously reported in an Asian and a European-American population.

La expresión del receptor del factor de crecimiento epidérmico (EGFR) en carcinomas mamarios hormonoindependientes / Expression of epidermal growth factor receptor (EGFR) in hormone dependent breast cancers

Objetivo. Estudiar la expresión del receptor del factor de crecimiento epidérmico (EGFR) en carcinomas ductal infiltrantes (CDI) hormonoindependientes así como sus posibles relaciones con otros parámetros clínico-biológicos. Material y método. El EGFR fue determinado, mediante un método de radioligando, en las membranas celulares de 115 CDI RE y RP negativos ( 7 % (p < 0,001); asimismo, fueron menos frecuentemente pS2 + (p < 0,001) y t-AP + (p: < 0,001). Pudimos seguir 70 pacientes (i: 54-168; mediana 85 meses) y observamos que los carcinomas EFGR + presentaron mayor número de recidivas (14/41 frente a 1/29; p: 0,002), pero no de muertes por el tumor. Conclusiones. 1) La positividad para el EGFR en los CDI RE y RP negativos se asoció con metástasis a distancia, mayor proliferación celular, menor positividad para pS2 y t-AP y con superior número de recidivas; 2) los mismos hallazgos, excepto el cambio en la proliferación celular, fueron constatados cuando el dintel de positividad fue establecido en 10 fmol/mg prot, y 3) El EGFR podría ser utilizado como factor pronóstico en este tipo de carcinomas mamarios

Objective. To determine the expression of epidermal growth factor receptor (EGFR) in ER-negative and Pg-R negative infiltrating ductal carcinomas of the breast (IDC) and to analyze the possible relationship between the EGFR positivity and some clinico-biological parameters of tumors. Material and methods. EGFR was measured by a single point radioligand assay in the cell surfaces of 115 ER-negative and Pg-R-negative ( 7 % (p 14 % (p: 0.077); likewise, they were lower frequently pS2-positive (p < 0.01) and t-PA-positive (p < 0.01). During the follow up time (median 85 months), the number of recurrences was higher in EGFR-positive than in EGFR- negative tumors (14/41 frente a 1/29; p: 0.002), but there was not differences in the number of deaths by the tumor. Conclusions. 1) The EGFR-positivity in ER-negative and PgR-negative IDCs is associated with distant metastasis, greater cellular proliferation (SP), lower positivity for pS2 and t-PA and greater number of recurrences. 2) Using 10 fmol/mg prot. as cut-off, we observed the same findings, except the change in cellular proliferation. 3) Our findings support the possible use of EGFR as a prognostic parameter in those breast carcinomas

pS2 citosólica y proliferación celular en carcinomas ductales infiltrantes de mama hormonodependientes / Cytosolic pS2 levels and cellular proliferation in ER-positive and PgR-positive infiltrating ductalcarcinomas of the breast

Objetivo: La proteína pS2 (TFF1/pS2) es una molécula inducida por los estrógenos en los carcinomas mamarios. Nosotros hemos querido estudiar su expresión en carcinomas ductales infiltrantes de mama RE + y RP + , correlacionarla con otros parámetros clínico-biológicos y conocer su impacto en la evolución. Material y metodos: La pS2 citosólica fue determinada utilizando un IRMA (CIS. BioInternational. Francia). Hemos analizado, además, las concentraciones citosólicas de catepsina D y activador del plasminógeno tipo tisular (t-AP), así como las del receptor del factor de crecimiento epidérmico (EGFR), oncoproteína erbB2, CD44v5 y CD44v6 en las membranas celulares. El tamaño tumoral, grado histológico (GH), afectación axilar, metástasis a distancia, ploidía, índice de DNA y fase de síntesis celular (FS) fueron también considerados. Resultados: Los tumores pS2-positivos (> 5ng/mg prot.) mostraron mayores concentraciones de catepsina D (p:0,0043) y t-AP (p:0,0089) y fueron, asimismo, menos frecuentemente GH3 (p:0,0231), FS > 7 % (p:0,0005) y FS > 14 % (p:0,0014). Durante el período de seguimiento (i: 1-147; 50,1+/­31,7; mediana 37 meses) los tumores pS2 positivos mostraron menor número de recidivas (5/101 vs 6/69; p:0,059), pero no de muertes por el tumor (1/101 vs 2/6); ns). Conclusiones: Nuestros resultados apoyan la relación inversa de la pS2 con la proliferación celular en los tumores RE+ y RP+, lo cual puede sugerir una nueva función biológica distinta a la de indicadora de la hormonodependencia. Sin embargo, necesitamos tener mayor tiempo de seguimiento para poder precisar si ello incide directamente en la evolución ulterior del tumor

Objective: The trefoil factor 1 (TFF1/pS2) is an estrogen-induced molecule in breast tumours. We wanted to study its expression in ER+ and PgR+ infiltrating ductal carcinomas of the breast (IDCs), and to correlate it with other clinical-biological parameters and the outcome. Material and methods: Cytosolic pS2 levels were measured using an IRMA (CIS. Biointernational. France) in 170 tumors. Likewise we determined the cytosolic levels of cathepsin D and tissue-type plasminogen activator (t-PA), as well as the concentrations of the epidermal growth factor receptor (EGFR), erbB2 oncoprotein, CD44v5 and CD44v6 on cell surfaces. Also the tumour size, histological grade (HG), axillary lymph node involvement, distant metastasis, ploidy, DNA index and of cellular synthesis phase (SP) was taken in account.Results: The pS2-positive (> 5 ng(/mg prot.) tumours showed higher concentrations of cathepsin D (p: 0.0043) and t-PA (p: 0.0089) than the pS2-negative ones. Likewise, they were less frequently HG3 (p: 0.0231), SP > 7 % (p: 0.0005) and SP > 14 % (p:0.0014). During the follow-up time (r: 1-147; 50,1+/­31,7; median 37 months) the pS2-positive tumors showed a less number of recurrences (5/101 vs 6/69; p: 0.059) but not of deaths by the tumor (1/101 vs 2/69). Conclusions: These results support an inverse relationship between pS2 positivity and cellular proliferation in IDCs and suggest a new role of this protein (different of the hormonodependence) in the biology of these breast carcinomas, while further studies will be required to establish the impact of this finding on their outcome