Luis O Gonzalez

Salome Gonzalez-Reyes

Laura Marin

Lucia Gonzalez

José-Manuel Gonzales

Luz Lamelas

Antonio M Merino

Ivan Pidal

Elena Alvarez

Alejandro Andicoechea

Luis O Gonzalez

Luis O Gonzalez

Luis O González

González-Reyes Salomé

Marín Laura

González Lucía

José M González

Maria L Lamelas

Alvarez Elena

Pidal Iván

Del Casar

Andicoechea Alejandro

Dr Francisco Vizoso

COMPARATIVE ANALYSIS AND CLINICAL VALUE OF THE EXPRESSION OF METALLOPROTEASES AND THEIR INHIBITORS BY INTRATUMOR STROMAL MONONUCLEAR INFLAMMATORY CELLS AND THOSE AT THE INVASIVE FRONT OF BREAST CARCINOMAS

Keywords: Peritumor stroma, MMP, TIMP, inflammation, leukocytes Peritumor stroma, MMP, TIMP, leukocytes, inflammation, tumor heterogeneity

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Tumors are composed not only of cancer cells but also of other cell types, notably stromal cells. The term stromal cells include cancer associated fibroblasts, endothelial cells, pericytes, and a variable representation of leukocytes. Leucocytes can account for as much 50% of the total tumor mass in invasive breast carcinomas. Historically, tumorinfiltrating leukocytes have been considered to be manifestations of an intrinsic defense mechanism against the development of tumors [START_REF] Johnson | Tumor immunology: Paul Ehrlich's heritage[END_REF][START_REF] Lin | Role of infiltrated leucocytes in tumour growth and spread[END_REF]. However, increasing evidence indicates that leukocyte infiltration can instigate a tumor phenotype, as evidenced by the presence of angiogenesis, tumor cell growth, and invasion [START_REF] Coussens | Inflammation and cancer[END_REF][START_REF] Daniel | CD4+ T cell-mediated antigen-specific immunotherapy in a mouse model of cervical cancer[END_REF][START_REF] Adams | Non-tolerance and autoantibodies to a transgenic self antigen expressed in pancreatic beta cells[END_REF]. This may be due to the fact that inflammatory cells probably influence cancer promotion by secreting cytokines, growth factors, chemokines and proteases, which in turn stimulate proliferation and invasiveness of/by cancer cells [START_REF] Sica | Altered macrophage differentiation and immune dysfunction in tumor development[END_REF][START_REF] Balkwill | Cancer and the chemokine network[END_REF][START_REF] Bitoux | Tumor-host interactions: the role of inflammation[END_REF]. In addition, accumulating clinical data for solid tumors show a correlation of high-density leukocytic infiltration into tumors with poor patient outcome [START_REF] Coussens | Inflammation and cancer[END_REF][START_REF] Bingle | The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies[END_REF][START_REF] Taskinen | Prognostic influence of tumor-infiltrating mast cells in patients with follicular lymphoma treated with rituximab and CHOP[END_REF][START_REF] Nonomura | Decreased number of mast cells infiltrating into needle biopsy specimens leads to a better prognosis of prostate cancer[END_REF]. Nevertheless, the prognostic significance of lymphoid infiltrates at the tumor site remains controversial, perhaps because the criteria of evaluation of tumor infiltrates are not sufficiently standardized to yield reliable and reproductible results. A typical leukocyte infiltrate is made up by a variable representation of leukocytes, including macrophages, neutrophils, mast cells, and B and T lymphocytes [START_REF] Coussens | Inflammation and cancer[END_REF][START_REF] Lin | Macrophages: modulators of breast cancer progression[END_REF]. Thus, there are evidences indicating that different types of leukocyte infiltrates occur in different breast carcinomas, and the type of infiltrate is probably related to de degree of tumor dissemination [START_REF] Canevari | 1975-1995 revised anti-cancer serological response: biological significance and clinical implications[END_REF].

The functional roles of the various leukocyte components of any tumor infiltrate remain to be fully elucidated, but evidence suggests that it may differ among tumors. Consequently, it is necessary to obtain biomarkers to assess the biological heterogeneity of these leukocytes infiltrates. Among possible biological markers metalloproteases (MMPs) could be good candidates worth studying. Indeed, MMPs play an essential role in the degradation of stromal connective tissue and basement membrane components, both of which are key elements in tumor invasion and metastasis. In addition, MMPs are able to impact in vivo on tumor cell behaviour as a consequence of their capacity to cleave growth factors, cell surface receptors, cell adhesion molecules, and chemokines/cytokines [START_REF] Egeblad | New functions for the matrix metalloproteinases in cancer progression[END_REF][START_REF] Noe | Release of an invasion promoter Ecadherin fragment by matrilysin and stromelysin-1[END_REF][START_REF] Manes | The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells[END_REF]. Furthermore, by cleaving proapoptotic factors, MMPs produce a more aggressive phenotype via generation of apoptotic resistant cells [START_REF] Fingleton | Matrilysin [MMP-7] expression selects for cells with reduced sensitivity to apoptosis[END_REF]. Also, MMPs positively regulate cancer-related angiogenesis, through their ability to [START_REF] Stetler-Stevenson | Matrix metalloproteinases in angiogenesis: a moving target for therapeutic intervention[END_REF], and negatively via the generation of angiogenesis inhibitors, such as angiostatin and endostatin, that are cleaved from large protein precursors [START_REF] Cornelius | Matrix metalloproteinases generate angiostatin: effects on neovascularization[END_REF] On the other hand, it is now accepted that tissue inhibitors of metalloproteases (TIMPs) are multifactorial proteins also involved in the induction of proliferation and the inhibition of apoptosis [START_REF] Jiang | Complex roles of tissue inhibitors of metalloproteinases in cancer[END_REF][START_REF] Wurtz | Tissue inhibitor of metalloproteinases-1 in breast cancer[END_REF]. Recently, we identified a phenotype of mononuclear inflammatory cells (MICs) at the intratumor stroma of breast carcinomas, which is associated with the development of distant metastasis. These MICs were characterized by overexpression of MMP-7, 9, 11, 13 and 14, as well as of TIMP-1 and 2 [START_REF] Gonzalez | Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse[END_REF] The clinical relevance of the expression of MMPs and TIMPs by intratumor stromal MICs led us to consider the potential biological and clinical significance of the expression of this enzymatic system by the MICs located at the invasive front of breast carcinomas. We focussed on the invasive front because it is the area where some of the most important interactions between cancer cells and the tumor supporting stroma take place [START_REF] Giatromanolaki | Tumour angiogenesis: vascular growth and survival[END_REF] . Therefore, the pattern of MMPs/TIMPs expression by MICs at the invasive front could reflect prognosis better than other tumor areas or, even, provide useful complementary information of clinical interest. Consequently, the aims of the present work were to compare the expression of MMPs and TIMPs by stromal MICs (T lymphocytes, B lymphocytes and macrophages), at two different locations of breast carcinomas: intratumoral and at the invasive front of breast carcinomas. We also studied their relationship with clinico-pathological characteristics and the prognosis. Our results demonstrate different patterns of expression of MMPs and TIMPs depending on MIC location within the tumor. We propose that the combined evaluation of these variable may provide a highly predictive tool for distant metastases.

MATERIALS AND METHODS

Patient selection, characteristics and tissue specimen handling

This study comprises 124 women with a histologically confirmed diagnosis of early invasive breast cancer (without distant metastasis at the time of the initial diagnosis) and treated between 1990 and 2003. We selected women with the following inclusion criteria: invasive ductal carcinoma, at least six histopathologically assessed axillary lymph nodes, and a minimum of five years of follow-up for those women without tumor recurrence. The exclusion criteria were the following: metastatic disease at presentation, prior history of any kind of malignant tumor, bilateral breast cancer at presentation, having received any type of neoadjuvant therapy, development of locoregional recurrence during the follow-up period, development of a second primary cancer, and absence of sufficient tissue in the paraffin blocks used for manufacturing the TAs [START_REF] Mcshane | Reporting recommendations for tumor marker prognostic studies[END_REF]. From patients fulfilling these criteria, we randomly selected a sample size of 124 patients according to four different groups with similar size and stratified with regard to nodal status and to the development of metastatic disease, which were the key measure variables of the study. Thus, we included an important number of events in both node-positive and node-negative patient subgroups (half of the cases that developed distant metastases during the follow-up period are included in each subgroup) in order to guarantee the statistical power of the survival analysis. Patient characteristics included in the two main groups, with or without distant metastases, are listed in Table 1. Tumor border configuration is classified as expansive(smooth, pushing border) versus infiltrative ( Dissection of adipose tissue by small glands or irregular cords or clusters of tumor cells) and is assessed on low-power examination.

Tumors were classified as negative for necrosis if there was not necrosis at all in the microscopic sections evaluated. When we recognized one o more focus of necrosis (independent of their size) we classified the tumor as positive for necrosis.

A desmoplastic reaction was defined as the pervasive growth of dense fibrous tissue around the tumor. The fibrous peritumoral stroma undergoes proliferation of newly formed fibroblasts in an edematous, myxomatous or highly collagenized matrix. One tumor was considered positive for peritumoral inflamammation if we observed a dense chronic inflammatory infiltrate rich in plasma cells and lymphocytes in at least 50% of Patients underwent either modified radical mastectomy or wide resection with axillary lymphadenectomy. Postoperative radiotherapy was given to 47 patients (42.7%). The criteria for systemic adjuvant therapy were as follows: i) node-negative patients with ER and /or PgR positive tumors received tamoxifen (20 mg per day during five years); ii) node-negative patients with ER and PgR negative tumors received six cycles of intravenous CMF (cyclophosphamide, methotrexate and 5-fluorouracil) every 3 weeks, if their tumors were either larger than one centimeter, moderately or poorly differentiated, or if patients were younger than 35 years old; iii) node-positive patients received six cycles of intravenous FEC (5-fluorouracil, epirubicin and cyclophosphamide) every 3 weeks, plus sequential tamoxifen if they had ER and/or PgRpositive tumors. Overall, 47 patients received chemotherapy, 39 patients received tamoxifen, and 17 patients received both types of systemic therapy.

Women were treated according to the guidelines used in our institution. The study adhered to national regulations and was approved by our Institution "Ethics and Investigation Committee". The end-point was distant metastatic relapse. The median follow-up period in patients without metastases was 85 months, and 46 months in patients with metastases. In addition, in the present study we analyzed the expression of the factors in normal mammary tissues obtained from 4 women that underwent cytoreductive surgery of the breast (age range: 35-60 years).

Tissue arrays and immunohistochemistry

Breast carcinoma tissue samples were obtained at the time of surgery. Routinely fixed (overnight in 10% buffered formalin), paraffin-embedded tumor samples stored in our pathology laboratories were used. Histopathologically representative tumor areas were defined on haematoxylin and eosin-stained sections and marked on the slide. Tumor tissue array (TA) blocks were obtained by punching a tissue cylinder (core) with a F o r P e e r R e v i e w 7 diameter of 1.5 mm through a histologically representative area of each 'donor' tumor block, which was then inserted into an empty 'recipient' tissue array paraffin block using a manual tissue arrayer (Beecker Instruments, Sun Praerie, WI, USA) as described elsewhere [START_REF] Parker | Assessment of interlaboratory variation in the immunohistochemical determination of estrogen receptor status using a breast cancer tissue microarray[END_REF]. Collection of tissue cores was carried out under highly controlled conditions. A total of four cores were used for each case. Two of these cores in each case corresponded to the tumor central area, and the other two cores corresponded to the invasive front. This method, evaluating two cores (double redundancy) of each tumor area has been shown to correlate well with conventional immunohistochemical staining [START_REF] Gonzalez | Study of matrix metalloproteinases and their tissular inhibitors in ductal "in situ" carcinomas of the breast[END_REF]. The invasive front was defined as the tumoral advancing edge. This corresponds to a 2 mm margin surrounding the tumor and containing cancerous cells. From the 124 tumor samples available, four TA blocks were prepared, each one containing 31 primary tumor samples, as well as external controls including four normal breast tissue samples from two healthy women who underwent reductive mammary surgery. These latter samples contained epithelial components on which immunostaining was not seen with any of the antibodies used.

Serial 5-µm sections of the high-density TA blocks were consecutively cut with a microtome (Leica Microsystems GmbH, Wetzlar, Germany) and transferred to adhesive-coated slide. One section from each tissue array block was stained with H&E, and these slides were then reviewed to confirm that the sample was representative of the original tumor. Immunohistochemistry was done on these sections of TA fixed in 10% buffered formalin and embedded in paraffin using a TechMate TM50 autostainer (Dako, Glostrup, Denmark). Antibodies for MMPs and TIMPs were obtained from Neomarker (Lab Vision Corporation, Fremont, CA, USA). The dilution for each antibody was established based on negative and positive controls (1/50 for MMP-2, -7, -14 and TIMP-2; 1/100 for MMP-9, -13, TIMP-1 and -3; and 1/200 for MMP-1, -11). The negative control was DakoCytomation mouse serum diluted to the same mouse IgG concentration as the primary antibody. All the dilutions were made in Antibody Diluent, (Dako, Glostrup, Denmark) and incubated for 30 min. at room temperature. As positive controls, breast tumor cytosol samples in which we confirmed the presence of the evaluated proteins by Western blot analysis were used as positive controls as shown previously [START_REF] Gonzalez | Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse[END_REF][START_REF] Gonzalez | Expression and prognostic significance of metalloproteases and their inhibitors in both luminal aand basal-like phenotypes of breast carcinomas[END_REF]. A single band of the expected molecular mass was observed for each protein [START_REF] Gonzalez | Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse[END_REF]. We also used other antibodies recognizing several factors, such as Tissue sections were deparaffinized in xylene and then rehydrated in graded concentrations of ethyl alcohol (100%, 96%, 80%, 70%, then water). To enhance antigen retrieval only for some antibodies, TA sections were microwave treated in a H2800 Microwave Processor (EBSciences, East Granby, CT, USA) in citrate buffer (Target Retrieval Solution; Dako) at 99ºC for 16min. Endogenous peroxidase activity was blocked by incubating the slides in peroxidase-blocking solution (Dako) for 5 min.

The EnVision Detection Kit (Dako) was used as the reactivity detection system.

Sections were counterstained with haematoxylin, dehydrated with ethanol and permanently coverslipped.

For each antibody preparation studied, the location of immunoreactivity, percentage of reactive area and intensity were determined. All the cases were semiquantified for each protein-stained area. An image analysis system with the Olympus BX51 microscope and soft analysis (analySIS ® , Soft imaging system, Münster, Germany) were used as follows: tumor sections were stained with antibodies according to the method explained above and counterstained with haematoxylin. There were different optical thresholds for both stains. Each core was scanned with a 400X power objective in two fields per core. Fields were selected searching for the protein-reactive areas. The computer program selected and traced a line around antibody-reactive areas (higher optical threshold: red spots), with the remaining, non-stained areas (haematoxylinstained tissue with lower optical threshold) standing out as a blue background. Any field had an area ratio of stained (red) versus non-stained (blue). A final area ratio was obtained after averaging two fields. To evaluate immunostaining intensity we used a numeric score ranging from 0 to 3, reflecting the intensity as follows: 0, no reactivity; 1, weak reactivity; 2, moderate reactivity; and 3, intense reactivity. Using an Excel spreadsheet, the mean score was obtained by multiplying the intensity score (I) by the percentage of reactivity area (PA) and the results were added together (total score: I x PA). This overall score was then averaged with the number of cores that were done for each patient. If there was no tumor in a particular core, then no score was given. In addition, for each tumor the mean score of two core biopsy samples was calculated. .

This scoring evaluation was based on a global evaluation of staining areas

Statistical analysis

Differences in percentages were calculated with the chi-square test. We consider cases with concordance between tumoral center and invasive front for each MMP or TIMP expression when in these cases one cellular type was positive or negative for the corresponding factor in both tumoral localizations. Immunostaining score values for each protein were expressed as a median (range). Correlation between score values was calculated by using the Spearman correlation test. Comparison of immunostaining values between groups was made with the Mann-Whitney or Kruskall-Wallis tests.

Statistical results were corrected applying Bonferroni's correction. For metastasis-free survival analysis we used the Cox's univariate method. Cox's regression model was used to examine interactions of different prognostic factors in a multivariate analysis. In the multivariate analysis only parameters that achieve statistical significance for distant relapse-free survival in the univariate analysis (as well as the type of systemic therapy) were included. Expression profiles were analyzed by an unsupervised hierarchical clustering method that¸ based on their similarity, organizes proteins in a tree structure.

Data was reformatted as follows: "-3" designated negative staining, "3" positive staining, and missing data was left blank. We used the Cluster 3.0 program (average linkage, uncentered correlation). Results were displayed with the Treeview program [START_REF] Eisen | Cluster analysis and display of genome-wide expression patterns[END_REF] TIMPs, between the tumor center and the whole tumor section in invasive breast carcinomas [START_REF] Vizoso | Study of matrix metalloproteinases and their inhibitors in breast cancer[END_REF]. while MICs at the invasive front showed a higher expression MMP-9 (p=0.016), MMP-11 (p<0.0001), and TIMP-2 (p=0.013). However, there were no significant differences in the expression of MMP-1, -2 and -13, and TIMP-1 between MICs of these two paired sets of tissue samples.

We analyzed the expressions of these factors in normal mammary tissues which were obtained from 4 women that underwent cytoreductive surgery of the breast. MICs from these normal tissue samples were negative for MMP-2, 7, 9, 11, 13 and 14; TIMP-1, 2 and 3. Only one of the 4 cases showed a weak staining for MMP-1 in a limited population of MICs.

The concordance between expressions of factors by intratumor MICs and by those at the invasive front, were of 59.6% for MMP- We also compared the expression of MMPs and TIMPs by these two MICs populations using the global immunohistochemical staining (score values) at the tumor center (Table 3). In this tumor area, , these factors were predominantly expressed, not only by MICs but also by cancer cells and, in a significant percentage, by fibroblasts, such as previously was reported by our team [START_REF] Gonzalez | Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse[END_REF][START_REF] Vizoso | Study of matrix metalloproteinases and their inhibitors in breast cancer[END_REF]. Our results demonstrate that the expression of each protein by intratumor MICs was significantly and positively associated with their corresponding score value (p<0.005, for MMP-7 and TIMP-3; and p<0.0001, for MMP-1, 9, 11, 13, and 14, and TIMP-2). Expressions of MMPs and

TIMPs by MICs at the invasive front showed significant associations with their corresponding score values for MMP-1, 11 and 13, and TIMP-2 (p<0.05, for each one of these comparisons), and for TIMP-1 (p<0.005). However, expression of MMP-2, 7 and 14, and TIMP-3, by MICs at the invasive front did not show significant associations with their corresponding score value (Table 3).

We also examined the possible relationship between the expression of MMPs or TIMPs by MICs, at the intratumor stroma (Table 4A) or at the invasive front (Table 4B), and the clinico-pathological characteristics of both patients and tumors. As shown in demonstrated that expression of MMP-11, 13, or 14, and TIMP-2 by these cells, was also significantly and independently associated with a higher probability of shortened distant relapse-free survival (Table 5).

To identify specific groups of tumors with distinct MMP/TIMP immunohistochemical expression profiles as well as their possible prognostic importance, the obtained data were evaluated by unsupervised hierarchical cluster analysis for each cellular type. This algorithm placed proteins on the horizontal axis and samples on the vertical axis based on similarity of their expression profiles. It produced a dendogram with well-defined cluster of cases for MICs at the center of the tumor as well as for MICs at the invasive front. Therefore, the dendogram showed a first-order division of the tumors into distinct MMP/TIMP molecular profiles, two distinct groups for MICs at the center of the tumor (with high or low MMP/TIMP profile, designated as group 1A -n=33-and group 2A -n=91-, respectively) (Figure 2a) and three distinct groups for MICs at the invasive front (with high, intermediate and low MMP/TIMP profiles, designated as group 1B -n=32-, group 2B -n=44-and group 3B -n=48-, respectively) (Figure 2b). MMP-1, 7, 9, 11, 13 and 14, and TIMP-1, 2 and 3, were identified as showing a significantly higher expression in groups 1A and 1B, compared with groups 2A and 3B, respectively (in both MICs populations). In addition, MMP-

DISCUSSION

This is the first study comparing the expression of MMPs and TIMPs by stromal

MICs located at both the intratumor area and at the invasive front of breast carcinomas.

Our results demonstrate in a significant number of cases, differences in the expression of these biological factors between intratumor and invasive front MICs. In addition, we have found that those different MIC populations might reflect different biological tumor behaviours and, thus, reinforce complementary clinical information in breast cancer patients.

The inflammatory response to tumor growth may be induced by at least two mechanisms. On the one hand, tumor growth and invasion are associated with significant cell death, some of which occurs by apoptosis or necrosis. It is well known that necrotic cells release numerous mediators of inflammation which are a potent stimulator of macrophages [START_REF] Ghersi-Egea | Rapid distribution of intraventricularly administered sucrose into cerebrospinal fluid cisterns via subarachnoid velae in rat[END_REF]. On the other hand, tumor cells may induce an inflammatory response by secreting cytokines and chemokines that activate macrophages and mast cells and recruit monocytes and lymphocytes [START_REF] Bitoux | Tumor-host interactions: the role of inflammation[END_REF]. In any case, examination of primary tumors frequently reveals an inflammatory infiltrate that varies in composition and intensity [START_REF] Brigati | Tumors and inflammatory infiltrates: friends or foes?[END_REF]. In addition, the functional role of the cellular components of the inflammatory infiltrates in tumors remains to be fully elucidated, but evidence suggests that it may differ from that observed in the context of physiological tissue repair [START_REF] Bitoux | Tumor-host interactions: the role of inflammation[END_REF]. Thus, although infiltrates of MICs in the neoplastic microenvironment may be expected to be beneficial for cancer patients, yet it often fails to correlate with good prognosis. Consequently with this latter hypothesis, our results suggest that inflammation can be both cancer stimulatory and cancer inhibitory, depending on the functional status of the infiltrating cell populations. Our results are in accordance with evidences indicating that MICs can express and secrete a variety of MMPs that together have the potential to hydrolyse all known ECM proteins [START_REF] Van Kempen | Noise exposure and children's blood pressure and heart rate: the RANCH project[END_REF]. In the tumor context, it reduces the physical barriers that tumoral cells have to overcome as they migrate towards lymphatic and blood vessels. In addition, some of the ECM protein degradation products display bioactivity on their own. Indeed, it has been reported that the release of cryptic fragments of laminin as a result of MMPmediated cleavage of laminin-5 γ2 chains that mimic EGF receptor (EGFR) ligands, and induce migration and invasive potential in EGFR expressing cells [START_REF] Schenk | Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution[END_REF]. In addition, it has been reported that leukocyte-derived MMP-7 cleaves and activates cell surface proheparin-binding EGF into its bioactive form, which in turn enhances tumor cell migration and survival [START_REF] Li | Calcium channel alpha2delta1 subunit mediates spinal hyperexcitability in pain modulation[END_REF]. Likewise, MMP-7 can cleave E-cadherin, facilitating tumor cell disaggregation and enhancing motility [START_REF] Rakha | Prognostic markers in triple-negative breast cancer[END_REF]. Therefore, by recruiting and harnessing the inflammatory response, weakly invasive tumors cells may acquire the means to disseminate.

All of these findings led us to formulate the question of whether inflammation and lymphocytic infiltration occur in order to favour the antitumor response to breast cancer or not. Our data suggest that intratumor leukocytes from peripheral blood suffer a modification of their phenotype that allow them to penetrate the tumor architecture, from the invasive front to the tumor center. This seem to be a dynamic processes in which inflammatory cells and immunomodulatory mediators present in the tumor microenvironment polarize host immune response toward specific phenotypes impacting tumor progression. Our results demonstrate biological heterogeneity among tumors with regard to these cellular infiltrates, identifying subsets of MICs, both at tumor center and at invasive front, which seem associated with the occurrence of distant

  area. For ER and PgR evaluations were used mouse anti-ER clone 1D5 at a dilution of 1/50, and anti-PR clone PgR 636 at a dilution of 1/50 (Dako, Glostrup, Denmark). Staining for ERs and PgRs was scored according to the method described by Allred et al.[START_REF] Allred | Prognostic and predictive factors in breast cancer by immunohistochemical analysis[END_REF].

8 MMP- 2 (

 82 policlonal, 1/50, Abcam Cambridge UK.), MMP-13 (clone 181-15A12, 1/100, Calbiochem), MMP-11 (clone SC3-05, 1/100, MERCK KgaA Darmstadt Germany).

  cells as well as to stromal cells. Nevertheless, in the present work we also evaluated the immunohistochemical staining exclusively on cancerous cells or on stromal MICs. Each evaluated field (400X power objective) contained at least ten stromal MICs. We distinguished stromal cells from cancer cells because the latter are larger in size. In addition, fibroblasts are spindle shaped whereas mononuclear inflammatory cells are rounded. On the other hand, while cancer cells are arranged forming either acinar or trabecular patterns, stromal cells are spreaded. Moreover, we used several markers to distinguish mononuclear inflammatory cells (T lymphocites (CD 3 , CD 45Ro,CD 4,CD 8) , B lymphocites (CD 20,CD 79a) and macrophages (CD68 ), all of them from Dako (Glostrup, Denmark).

Figure 1

 1 Figure 1 shows representative examples of MMPs and TIMPs expression by MICs at the center of the tumor and at the invasive front of breast carcinomas. Immunostaining for these proteins shows a cytoplasmic location both in positive cancerous cells as in positive MICs. With regard to MMP-14 expression, it is noteworthy the positive immunostaining at both cytoplasmic and membrane locations. At least 70% of MICs positives for each MMPs or TIMPs showed a positive immunostaining at each evaluated field. As Table 2 shows, the expression of MMPs and TIMPs by MICs varied among tumors. MICs at the tumor center showed MMP-7 (p<0.0001), MMP-14 (p<0.0001), and TIMP-3 (p<0.0001), positive staining than MICs at the invasive front;

  Our results show levels of discordance in the expressions of MMPs and TIMPs by intratumor MICs and by those located at the invasive front, ranging from 2.5% for MMP-2 to 54.4% for TIMP-3. Likewise, clustering analysis shows two different groups, with low or high MMP/TIMP molecular profile, in both MICs populations, but each one of them with non-identical MMP/TIMP patterns. In addition, this latter analysis led us to identify a third group of tumors, which correspond to these cases with MICs showing an intermediate MMP/TIMP molecular profile at invasive front. Therefore, these findings led us to consider the existence of functional differences in host MICs in these in a significant percentage of cases, which could represent a new contribution to the knowledge of the stromal heterogeneity of breast carcinomas.Intratumor MICs showed a positive expression of MMP-7 and 14, and TIMP-3 more frequently than MICs at the invasive front. `However this latter population of MICs showed more frequent expressions of MMP-9 and 11, and TIMP-2. This different pattern of expression of MMPs and TIMPs may correspond to differences in cellular density, which is higher in the tumor center, and/or to different biological mechanisms of interaction between cancer cells and MICs in those two different tumor areas.Nevertheless, it is noteworthy that in many tumors there was a lack of concordance in the expression of MMPs or TIMPs by cancer cells and MICs, which seems to indicate that these cell types display an independent phenotype with regard to expression of those enzymes. It was also remarkable that, except for MMP-2 and TIMP-1, there were significant and positive relationships between the expression of each MMP/TIMP by intratumoral MICs and their corresponding global expression (score values) in the tumor center; whereas expression of MMPs/TIMPs by MICs at the invasive front only showed significant, but lower, relationships with score values in the tumor center for MMP-1, 11 and 13, and TIMP-1, and 2. Likewise, we found low percentages of concordance in MMPs/TIMPs expression between MICs and cancer cells, both at the tumor center and at the invasive front. All of these findings add up to the accumulating evidence of the presence of different phenotypes of MICs infiltrating breast carcinomas at two differentiated tumor areas: tumor center and invasive front. Our results also show several significant associations between MMPs/TIMPs expression by MICs with clinico-pathological factors indicative of tumor progression, depending of the tumoral area analyzed. With regard to prognosis, we found a significant value of MMP-11, 13 and 14, and TIMP-2, by MICs both at the tumor center and at the invasive front, as independent predictive factors of distant metastases. However, MMP-9 and TIMP-2 expression by MICs at the center of the tumor, and not at invasive front, were associated with distant metastasis. As of today, we do not have a reasonable explanation for the prognostic significance of the different MIC locations. Even so, our data suggested that host stromal MICs that appear at sites of active tumor invasion may have a different activation status of biological relevance for tumor growth and progression. Likewise, it was remarkable our finding indicating a high prognostic value of the combination of several molecular profiles of MMP/TIMP on clustering analysis, of each MICs population. Patients with high MMP/TIMP patterns in the corresponding MICs populations at the center of the tumor, as well as at the invasive front, had the highest probability of distant metastases. On the contrary, patients with low MMP/TIMP patterns in both MICs populations had the lowest risk of distant metastases. Therefore, our results indicate the importance of evaluating the expression of these factors, that are involved in tumor growth, by MICs on a tumor location basis, thus providing a valuable complementary information on tumor behavior.

  , our findings are in accordance with these of other authors indicating that leukocyte infiltration can promote tumoral phenotypes, such as angiogenesis, growth, and invasion[START_REF] Daniel | CD4+ T cell-mediated antigen-specific immunotherapy in a mouse model of cervical cancer[END_REF][START_REF] Adams | Non-tolerance and autoantibodies to a transgenic self antigen expressed in pancreatic beta cells[END_REF][START_REF] Cabernard | Results of the modified dacryocystorhinostomie described by Werb (author's transl)[END_REF].In summary, our results reveal dynamic and reciprocal interactions between cancer cells and cells of the tumor microenvironment which may orchestrate events critical to tumor evolution towards metastasis. Likewise, our data may contribute to identify subsets of MICs and protein elements of the microenvironment as attractive targets for therapeutic strategies.

Figure 1 .Figure2:Figure 3 .

 13 Figure 1. (400x). Comparative expresión of MMPs and TIMPs by MICs (arrows) in the center (left) of the tumor and at the invasive front (right) of breast carcinoma. a-Positive expression of MMP-7; b-No expression of MMP-7;c-Positive expression of MMP-14; d-No expression of MMP-14; e-Positive expression of TIMP-3; f-No expression of TIMP-3; g-No expression of MMP-9; h-Positive expression of MMP-9; i-No expression of MMP-11; j-Positive expression of MMP-11; k-No expression of TIMP-2; l-Positive expression of TIMP-2.

  2% for MMP-9, 66.7% for MMP-11, 67.5% for MMP-13, 54.2% for MMP-14, 65% for TIMP-1, 57.7% for TIMP-2, and 45.6% for TIMP-3.The concordances between the expression of MMPs and TIMPs by MICs and by cancer cells were also analyzed at the tumor center and at the invasive front. At tumor center, the results show the following expression concordances between the two cell types:

	11
	74.79% for MMP-1, 68.8% for MMP-2, 62.6% for MMP-7, 37.4% for MMP-9, 45.1% for
	MMP-11, 59.7% for MMP-13, 62.9% for MMP-14, 31.7% for TIMP-1, 48.8% for
	TIMP-2, and 68.5% for TIMP-3. At the invasive front, our results show the following
	accordances between the two cell types: 76.6% for MMP-1, 62.3% for MMP-2, 33.9%
	for MMP-7, 28.9% for MMP-9, 58% for MMP-11, 48.4% for MMP-13, 39.9% for F r o MMP-14, 43.8% for TIMP-1, 61.3% for TIMP-2, and 53.8% for TIMP-3.
	P
	e
	e r
	R
	e
	v i e
	w

1, 97.5% for MMP-2, 50.9% for MMP-7,

Table 4 (

 4 A and B), our results demonstrate a significant relationship of the expression of several MMPs or TIMPs by MICs at the tumoral center, with that at the invasive ER-negative status, desmoplastic reaction, peritumor inflammation or advanced edge tumor). Nevertheless, we also found a significant association of the expression some TIMPs by MICs at the invasive front with parameters indicatives of less tumoral aggressiveness. For instance, TIMP-1 expression was associated with negative nodal status and non-perineural infiltration, and TIMP-3 expression with negative nodal status (Table4A and B).We also analyzed the possible influence of single MMP or TIMP expression by MICs at the invasive front, or at the tumor center, on distant relapse-free survival. As shown in Table4, multivariate analysis showed that expressions of MMP-9, -11, -13, or -14, TIMP-1 or TIMP-2, by MICs at the tumor center, were significantly associated with a high rate of distant metastases. With regard to MICs at the invasive front, these results

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front, as well as with the clinico-pathological parameters indicatives of tumoral aggressiveness (such as younger age from patients, lymph node involvement, higher

TABLE 1

 1 Basal characteristics of 124 patients with invasive ductal carcinoma of the breast.

		WITHOUT	WITH RECURRENCE
	CHARACTERISTICS	RECURRENCE
		N (%)	N (%)
	TOTAL CASES
	AGE (YEARS)
	<57	28 (47.5)	38 (58.5)
	>57	31 (52.5)	27 (41.5)
	MENOPAUSAL STATUS
	PREMENOPAUSAL	18 (30.5)	18 (27.7)
	POSTMENOPAUSAL	41 (69.5)	47 (72.3)
	TUMORAL SIZE
	T1	31 (52.5)	27 (41.5)
	T2	28 (47.5)	38 (58.5)
	NODAL STATUS
	N-	28 (47.5)	28 (43.1)
	N+	31 (52.5)	37 (56.9)
	HISTOLOGICAL GRADE (SBR)
	I	20 (33.9)	14 (21.5)
	II	26 (44.1)	35 (53.8)
	III	13 (22)	16 (24.6)
	NOTTINGHAM PRONOSTIC INDEX
	<3.4	28 (47.5)	17 (26.2)
	3.4-5.4	24 (40.7)	35 (53.8)
	>5.4	7 (11.9)	13 (20)
	ESTROGEN RECEPTORS
	NEGATIVE	18 (30.5)	35 (53.8)
	POSITIVE	34 (57.6)	26 (40)
	PROGESTERONE RECEPTORS
	NEGATIVE	22 (37.3)	41 (63.1)
	POSITIVE	30 (50.8)	20 (30.8)
	ADJUVANT RADIOTHERAPY
	NO	44 (74.6)	33 (50.8)
	YES	15 (25.4)	32 (49.2)
	ADJUVANT SYSTEMIC THERAPY
	CHEMOTHERAPY	18 (30.5)	29 (44.6)
	ADJUVANT TAMOXIFEN	24 (40.7)	15 (23.1)
	CHEMOTHERAPY+SEQUENTIAL TAMOXIFEN	10 (16.9)	7 (10.8)
	NO TREATMENT	7 (11.9)	14 (21.5)

Table 2 .

 2 Expression of MMPs and TIMPs by monocytes at invasive front and at tumoral center of invasive ductal carcinomas of the breast.

		TUMORAL
	CENTER FACTOR N. POSITIVE	INVASIVE FRONT N .POSITIVE	p VALUE
		CASES (%)	CASES (%)
	MMP-1	81 (68.1)	87 (73.1)	N.S.
	MMP-2	2 (1.7)	1 (0.8)	N.S.
	MMP-7	60 (53.6)	17 (15.2)	0.0001
	MMP-9	13 (10.8)	28(23.3)	0.016
	MMP-11	39 (33.3)	66 (56.4)	0.0001.
	MMP-13	40 (33.3)	29 (24.2)	N.S.
	MMP-14	61 (51.7)	35 (29.7)	0.0001
	TIMP-1	33 (27.5)	47 (39.2)	N.S.
	TIMP-2	44 (37.3)	64 (54.2)	0.013
	TIMP-3	67 (54.5)	10 (8.1)	0.0001

TABLE 3

 3 Relationship between MMPs and TIMPs expressions by monocytes at tumoral center or at invasive front and global expressions of MMPs andTIMPs (score values) in tumoral center. Data are represented as median of score values (range).

	Columns represent tumors with monocytes with negative (-) v.s. positive (+)
	immunostaining for each factor.

TABLE 4A .

 4A Significant associations between expressions of MMP or TIMPs by monocytes at tumoral center and clinico-pathological characteristics from breast carcinomas.

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				Histopathology
	FACTORS AND CHARACTERISTICS
				N TOTAL OF CASES	N (%) OF POSITIVE CASES	P
				INVASIVE FRONT
	MMP-1
	AGE
			<57 >57		66 58		53 (80.3) 37 (63.8)	0.040
	MMP-7
	NODAL STATUS
			N-N+		55 66		13 (23.6) 6 (9.1)	0.029
	MMP-9
	PROGESTERONE RECEPTORS
		POSITIVE NEGATIVE		48 62		7 (14.6) 19 (30.6)	0.049
	TUMORAL NECROSIS
	YES NOT PERITUMORAL INFLAMATION MMP-11 F o YES NOT r ESTROGEN RECEPTORS	14 104 46 70		4 (28.5) 24 (23.1) 33 (71.7) 33 (47.1)	0.034 0.028
	MITOSIS	POSITIVE NEGATIVE	P	57 52		26 (45.6) 34 (65.4)	0.038
	MMP-13 NODAL STATUS	<10 >10 N-N+	e 65 52 e r 54 66	31 (47.7) 35 (67.3) 18 (33.3) 11 (16.7)	0.033 0.034
	TIMP-1 NODAL STATUS DESMOPLASTIC REACTION N-N+ YES NOT PERINEURAL INFILTRATION YES NOT SBR I		55 66 81 40 11 110 33	R e v i 29 (52.7) 18 (27.3) 26 (32.1) 21 (52.5) e 1 (9.1) 46 (41.8) w 13 (39.4)	0.004 0.003 0.034
			II		60		17 (28.3)	0.015
			III		28		17 (60.7)
	TIMP-2
	AGE
			<57 >57		64 55		41 (64.1) 24 (43.6)	0.026
	PERITUMORAL INFLAMATION
			YES NOT		46 69		33 (71.7) 30 (43.5)	0.010
	TIMP-3
	NODAL STATUS
			N-N+		55 68		10 (18.2) 0 (0)	0.0001
	MENOPAUSE
		PREMENOPAUSE POSTMENOPAUSE		36 87		0 (0) 10 (11.5)	0.034

TABLE 4B .

 4B Significant associations between expressions of MMP or TIMPs by monocytes at invasive front and clinico-pathological characteristics from breast carcinomas

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	Histopathology
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TABLE 5 :

 5 Cox's univariate(HR) and multivariate(RR) analysis of the relationship between MMPs and TIMPs expression and relapse-free survival in Monocytes. Abbreviations: HR, hazard ratio; RR, relative risk; CI,confidence interval; MIC, mononuclear inflammatory cells. *p<0.001; **p<0.005; ***p<0.01; ****p<0.05

				Event
		FACTOR	N of Patients	Frequenc	HR (95%CI)	RR (95%CI)
				y
	MMP-1	Monocytes (-)vs(+) at tumoral center	38/81	17/47	1.61 (0.92-2.8)
		Monocytes (-)vs(+)at invasive front	34/90	12/53	1.83 (0.97-3.43)
	MMP-2	Monocytes (-)vs(+) at tumoral center	120/2	64/1	0.78(0.1-5.64)
		Monocytes (-)vs(+) at invasive front	121/1	63/1	1.04 (0.14-7.61)
	MMP-7	Monocytes (-)vs(+) at tumoral center	54/61	25/38	1.47(0.88-2.44)
		Monocytes (-)vs(+) at invasive front	102/19	52/11	1.09 (0.59-2.11)
	MMP-9	Monocytes (-)vs(+) at tumoral center	109/14	50/14	3.4 (1.84-6.3)*	3.23(1.71-6.1)*
		Monocytes (-)vs(+) at invasive front	93/28	46/17	1.44 (0.82-2.51)
	MMP-11	Monocytes (-)vs(+) at tumoral center	82/40	25/39	5.41(3.21-9.1)*	5.18(3.02-8.87)*
		Monocytes (-)vs(+) at invasive front	53/66	16/47	3.36(1.89-5.95)*	3.02 (1.66-5.5)*
	MMP-13	Monocytes (-)vs(+)i at tumoral center	81/43	35/30	2.03(1.23-3.3)**	2.2(1.3-3.7)**
		Monocytes (-)vs(+) at invasive front	91/29	40/23	2.27(1.35-3.8)**	2.5(1.43-4.36)*
	MMP-14	Monocytes (-)vs(+) at tumoral center	58/66	16/49	4.4 (2.43-7.97)*	4.44(2.36-8.35)**
		Monocytes (-)vs(+) at invasive front	83/35	33/28	2.9(1.7-4.8)*	3.26(1.89-5.6)*
	TIMP-1	Monocytes (-)vs(+) at tumoral center	90/33	36/28	2.52(1.54-4.18)*	2.14(1.27-3.61)**
		Monocytes (-)vs(+) at invasive front	74/47	36/27	1.23 (0.74-2.04)
	TIMP-2	Monocytes (-)vs(+)i at tumoral center	76/47	24/40	3.78(2.26-6.3)*	4.3(2.5-7.38)*
		Monocytes (-)vs(+) at invasive front	54/65	20/41	2.01 (1.17-3.44)*** 2.25 (1.28-3.96)**
	TIMP-3	Monocytes (-)vs(+) at tumoral center	56/68	28/37	1.12(0.68-1.83)
		Monocytes (-)vs(+) at invasive front	113/10	56/8	1.94 (0.92-4.09)
	Cluster Group at tumoral center
		Group 1A vs. Group 2A	32/91	33/33	5 (3-8.5)	4.7 (2.7-8)*
	Cluster Group at invasive front
		Group 3B (low)	32	23	1	1
		Group 2B (intermediate)	44	25	2.7 (1.4-5.1)	1.6 (0.8-3.1)
		Group 1B (high)	48	17	1.8 (0.9-3.3)	2.2 (1.1-4.4)**
	Combination of Cluster Groups*
		Group 2A and Group 3B	44	14	1	1
		Group 2A and Group 1B or 2B	47	19	1.3 (0.6-2.6)	1.1 (0.5-2.3)
		Group 1A and Group 3B	3	3	5.5 (1.5-19.4)	9.9 (2.6-37.6)
		Group 1A and Group 1B or 2B	30	29	5.8 (3-11.3)	4.8 (2.4-9.5)*

Page 32 of 31 Published on behalf of the British Division of the International Academy of Pathology Histopathology

Published on behalf of the British Division of the International Academy of Pathology Histopathology

FIS-PI070306, Fondo de Inversión Sanitaria del Instituto

Carlos III (FIS-Spain), FICYT (IB08-170), and Obra Social Cajastur.


Raphaela ; Fritsche
email: raphaela.fritsche@charite.de

Andreas Grützkau

Aurelia Noske

Ingo Melcher

Klaus-Dieter Schaser

Peter M Schlag

Veit Hans-Udo U Kasper

Jonas Krenn

Christine Sers

Andreas Noske

Aurelia Melcher

Ingo Schaser

Klaus-Dieter Schlag

Peter Kasper

Raphaela Fritsche-Guenther

Andreas Gruetzkau

Aurelia Noske

Veit Krenn

Dr. rer. nat Raphaela Fritsche

Therapeutic potential of CAMPATH-1H in skeletal tumours

Keywords: CD52, skeletal tumours, complement-dependent cytotoxicity, osteosarcoma CD52, skeletal tumours, complement-dependent cytotoxicity, osteosarcoma

la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

detected the expression of the CD52 mRNA and protein both in vivo and in vitro. The malignant tumours showed a higher CD52 expression compared to the benign tumours, suggesting a role in the development and progression of bone tumours. Interestingly, immunohistochemistry and flow cytometry revealed that CD52 was not only expressed on the surface of the tumour cells, but also in the cytoplasm. Our results obtained in osteosarcoma cells show that CAMPATH-1H leads to a complement-independent reduction of viable cells. Conclusion: CD52 is expressed in a variety of bone tumours and the in vitro studies presented herein suggest that CAMPATH-1H treatment might have therapeutic potential for osteosarcoma patients with poor clinical prognosis.

Introduction

CD52 is a glycosylphosphatidylinositol (GPI)-anchored antigen, also called the CAMPATH-1H antigen. It is abundantly expressed on normal and malignant lymphocytes, such as cells from most B-and T-cell malignancies and in a minority of myeloid leukaemias on monocytes, macrophages and eosinophils [START_REF] Hale | The CAMPATH-1 antigen (CDw52)[END_REF][START_REF] Elsner | Surface and mRNA expression of the CD52 antigen by human eosinophils but not by neutrophils[END_REF] . In addition, CD52 can be found in the epithelial cells of the distal epididymis and the vas deferens and is acquired by sperm cells during their passage through the genital tract [START_REF] Kirchhoff | CD52 is the 'major maturation-associated' sperm membrane antigen[END_REF] . The structure of the CD52 protein is unusual. It is a very small, heavily glycosylated antigen with glycolipid-like properties. The sperm CD52 protein differs from the lymphocyte CD52 protein in its carbohydrate structure [START_REF] Schroter | Male-specific modification of human CD52[END_REF] . The physiological role of CD52 on lymphocytes is unclear. The high density of CD52 and the close proximity of the antibody binding site to the cell surface make it a particularly good target for therapeutic options [START_REF] Bindon | Importance of antigen specificity for complement-mediated lysis by monoclonal antibodies[END_REF] . These properties may contribute to the efficacy of the therapeutically used CD52 antibody, also called CAMPATH-1H (Alemtuzumab). CAMPATH-1H is capable of complementactivation and antibody-mediated cellular cytotoxicity leading to a depletion of lymphocytes [START_REF] Heit | Ex vivo T-cell depletion with the monoclonal antibody Campath-1 plus human complement effectively prevents acute graft-versus-host disease in allogeneic bone marrow transplantation[END_REF][START_REF] Xia | Efficient complement-mediated lysis of cells containing the CAMPATH-1 (CDw52) antigen[END_REF] . Stanglmaier et al. also suggested a caspase-independent form of apoptosis that may be induced by CAMPATH-1H in patients with chronic lymphocytic leukaemia [START_REF] Stanglmaier | Rituximab and alemtuzumab induce a nonclassic, caspase-independent apoptotic pathway in B-lymphoid cell lines and in chronic lymphocytic leukemia cells[END_REF] . At present, CD52-specific antibodies are used to treat lympho-proliferative disorders like B-cell chronic lymphocytic leukaemia, to deplete lymphocytes in bone marrow transplants, in the therapy of transplant rejection, to control graft versus host disease and, most recently, to treat Behcet's disease and multiple sclerosis [START_REF] Waldmann | Elimination of graft-versus-host disease by in-vitro depletion of alloreactive lymphocytes with a monoclonal rat anti-human lymphocyte antibody (CAMPATH-1)[END_REF][START_REF] Giralt | The role of alemtuzumab in nonmyeloablative hematopoietic transplantation[END_REF][START_REF] Magliocca | The evolving role of alemtuzumab (Campath-1H) for immunosuppressive therapy in organ transplantation[END_REF][START_REF] Cox | Lymphocyte homeostasis following therapeutic lymphocyte depletion in multiple sclerosis[END_REF][START_REF] Lockwood | Remission induction in Behcet's disease following lymphocyte depletion by the anti-CD52 antibody CAMPATH 1-H[END_REF][START_REF] Dearden | Alemtuzumab in lymphoproliferate disorders[END_REF] .

By microarray analysis, we found that CD52 mRNA is significantly overexpressed in giant cell tumours of the bone [START_REF] Guenther | Giant cell tumours of the bone: molecular profiling and expression analysis of Ephrin A1 receptor, Claudin 7, CD52, FGFR3 and AMFR[END_REF] . Further experiments revealed a significant upregulation of CD52 mRNA in chondrosarcoma and osteosarcoma tissue samples, suggesting a previously unknown deregulation of CD52 in bone malignancies.

Material and Methods

Tissue studies

Patients and tissues

Immunohistochemistry

Immunohistochemical analysis of CD52 was performed on 26 giant cell tumours (n=18 primary tumours, n=8 relapses), 14 osteosarcomas (n=7 conventional, n=7 metastases) and 6 chondrosarcomas on paraffin-embedded microsections. Three tissue microarrays of cartilage, bone and skeletal tumours were stained for CD52 expression. Tissues were cut into 1-3 µm sections and deparaffinised through serial dilutions of alcohol. Retrieval of the CD52 antigen was performed by incubation with proteinase K (DAKO) and 0.05% trypsine (Zymed). Anti-CD52 antibody (1:40 rat anti-human, Serotec), secondary antibody (1:40 rabbit anti-rat DAKO) and the APAAP complex (1:100 rat DAKO) were incubated for 30 min, 10 min and 10 min at room temperature, respectively. Fuchsin + (DAKO) was used as the chromogen. Nuclear counter staining was performed with haemalum. Negative control sections were not incubated with the primary antibody and in no case showed evidence of staining with colour reactions under microscopic examination.

The staining of all cases was semi-quantitatively scored by two different observers using the following method modified from Remmele and Stegner [START_REF] Remmele | Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue[END_REF] Aigner, Institute of Pathology, Friedrich-Alexander University Erlangen, Erlangen, Germany. For the establishment of a human giant cell tumour primary culture, resection tissue (male, 28 years) was minced in dispase-collagenase with PBS (phosphate buffered saline) and incubated for 20 min at 37°C. After that the cells were resuspended in Leibowitz L15 medium (Lonza) and, together with small tissue fragments, were then transferred to 75-cm² flasks for subsequent culturing. Half of the medium was changed on the second day. All cells were incubated in a humidified atmosphere of 5% CO 2 in air at 37°C.

Flow cytometry

Direct fluorescein isothiocyanate (FITC)-conjugated rat anti-human CD52 antibody

Proliferation assay

MNNG/HOS cells were plated in 96-well-plates and treated with 10-1,000 µg/ml CAMPATH-1H (Schering) for up to 96 h. For complement-mediated cytotoxicity, 10% human complement was added to the media. For the control, the complement was heatinactivated for 1 h at 56°C. Proliferation of cells was measured using the MTT cell proliferation kit from Roche.

Immunoblot

Protein extracts of MNNG/HOS cells were prepared by incubation with sodium dodecylsulfate (SDS)-cell lysis buffer (10% SDS, 1M Tris-HCl pH 7.5, EDTA 0.5 M pH 8). Reagents for SDS-PAGE and Western blotting were obtained from Bio-Rad Laboratories (Richmond, CA). Electrophoresis was performed and the proteins were transferred onto nitrocellulose membranes (Schleicher & Schüll). Unbound protein sites were blocked with 5% milk powder in Tris-buffered saline containing 0.01% Tween-20 (TBST). Thereafter, specific proteins were detected by incubation with mouse antihuman PARP (1:500 BD) and mouse anti-human actin (1:5,000 Chemicon) antibodies F o r P e e r R e v i e w 9 overnight at 4°C followed by HRP-conjugated rabbit anti-mouse (1:5,000 Dianova) secondary antibody. Blots were developed using the ECL system from Amersham.

Results

Tissue studies

Increased CD52 expression in bone malignancies

RNA derived from eight osteosarcoma samples (n=4 conventional, n=4 metastases) and from the human osteoblast primary culture HOBc was analysed using Affymetrix HG U133A microarrays (GEO accession number GSE14359; Guenther et al. submitted). A significantly higher expression of CD52 was found in osteosarcoma metastases as compared to conventional osteosarcoma (p=0.000619). CD52 mRNA levels were below background in HOBc cells and the conventional osteosarcomas, except for in the OS18 cell line (relative expression level of 1,022 and 1,336). In the microarray analysis of primary and recurrent giant cell tumours of bone, CD52 mRNA was lower in recurrent as compared to primary giant cell tumours [START_REF] Guenther | Giant cell tumours of the bone: molecular profiling and expression analysis of Ephrin A1 receptor, Claudin 7, CD52, FGFR3 and AMFR[END_REF] .

For the validation of microarray data, 14 osteosarcoma tissue samples (n=7 conventional and n=7 lung metastases) and 26 giant cell tumour tissue samples (n=18 primary, n=8 recurrence) were stained for CD52. As a control, a tissue microarray of non-neoplastic bone samples containing 4 foetal and 6 adult samples was used. High levels of CD52 protein were seen in foetal bone (Fig. 1A), while adult bone had lower CD52 levels (Fig. 1B). Interestingly, the osteoblasts in foetal bone samples showed the strongest CD52 staining, while the osteoblasts in adult bone were CD52 negative or showed only weak expression of CD52. We also found a largely cytoplasmic localisation, with only rare cases having a nuclear expression pattern of CD52 protein.

The osteocytes were CD52 positive in foetal and adult bone (Fig. 1A,B). The primary 1C). All ostoesarcoma samples showed a strong immunoreactivity for the CD52 antigen (IRS 6.1), but no significant differences could be observed between the conventional osteosarcomas and the metastases (p=0.515). The latter showed generally a lower CD52 expression (IRS=5.6) as compared to the conventional osteosarcomas (IRS=6.6). In most cases, the CD52 staining was cytoplasmic, but some nuclear localisation could also be observed (Fig. 1D).

CD52 protein was detected in 18/18 primary giant cell tumours and 8/8 recurrence samples. The macrophage-like cells and the multinucleated giant cells showed on average a moderate cytoplasmic staining intensity (Fig. 1E).

High expression of CD52 in chondrosarcoma and cartilage CD52 protein expression was also tested in non-neoplastic and neoplastic cartilage samples. The non-neoplastic tissue microarray included 4 embryonal, 6 foetal and 10 adult cartilage tissue samples, and the neoplastic tissue array had 6 chondrosarcoma tissue samples. Non-neoplastic cartilage was CD52 negative in all embryonal (Fig. 2A) and in 4/5 foetal cartilage samples (Fig. 2B). Only one foetal cartilage tissue had low CD52 expression. In contrast, CD52 expression could be observed in chondrocytes of adult cartilage by immunohistochemistry (Fig. 2C). Furthermore, a primary chondrocyte culture showed a low cytoplasmic CD52 expression (Fig. 2D). The chondrosarcoma tissue samples had the highest IRS (IRS 7.3) compared to osteosarcomas (IRS 6.1) and giant cell tumours (6.2). In total, 76-100% of the chondrosarcoma cells were positive (PP=3.8) with an average staining intensity of 2.1. The grade 2 chondrosarcomas showed a moderate cytoplasmic expression of CD52, whereas in the grade 3 samples a strong cytoplasmic CD52 staining could be observed (Fig. 2E). Because of the small

CD52 expression is rare in skeletal tumours

In addition to osteosarcoma, chondrosarcoma and giant cell tumours, benign bone-and cartilage-related, malignant fibrous and skeletal tumours with undefined origin (fibrous dysplasia and aneurysmal bone cyst) were analysed for CD52 expression. Results from immunohistochemical evaluation of all skeletal tumours are shown in Table 2.

Cytoplasmic and nuclear CD52 expression could be detected in 4/5 benign chondromas (Fig. 3A), 2/3 benign osteochondromas (Fig. 3B), 3/3 malignant fibrosarcomas (Fig. 3C), 3/3 benign aneurysmal bone cysts (Fig. 3D) and 5/5 benign osteomas (Fig. 3E).

Fibrous dysplasia stained weakly and CD52 protein was only detected in the stroma (Fig. 3F).

In vitro studies

Membranous and intracellular localisation of CD52

Immunohistochemical staining suggested a dual localisation of CD52 both on the surface of the cells, as expected for GPI-anchored glycoproteins, but also in the cytoplasm. To analyse the intra-and extracellular expression of CD52, flow cytometric analysis was performed using the CD52 FITC-conjugated antibody in osteosarcoma and chondrosarcoma cell lines, giant cell tumour and chondrocyte and osteoblast primary cultures (Table 3, Fig. 4). The human T-cell lymphoma cell line Hut78 was included as a positive control due to known expression of CD52 [START_REF] Rebello | Pharmacokinetics of CAMPATH-1H: assay development and validation[END_REF] .

Staining of Hut78 for membranous and intracellular CD52 revealed 32% and 93% positive cells, respectively (Fig. 4A). In MNNG/HOS osteosarcoma cells, 10% of cells showed a membranous staining, while 69% showed an intracellular staining (Fig. ). The chondrosarcoma cell line AD contained 74% positive cells, the highest proportion of CD52 positive cells (Fig. 4E). The highest membranous expression could be detected in the giant cell tumour primary culture, with 20% positive stained cells (Fig. 4F).

CAMPATH-1H inhibits growth of osteosarcoma cells

The therapeutic CD52 antibody CAMPATH-1H was shown to deplete lymphocytes through antibody-dependent cytotoxicity or complement-dependent cytotoxicity. To study the effect of the CAMPATH-1H antibody on bone tumours, we incubated the human osteosarcoma cell line MNNG/HOS with 50 µg/ml or 100 µg/ml CAMPATH-1H and assayed for cell growth.

Using 50 µg/ml and 100 µg/ml CAMPATH-1H in media containing 10% human complement, a significant reduction of proliferating MNNG/HOS cells was observed when compared to cells incubated with inactive complement (50 µg/ml p=0.026, 100 µg/ml p=0.021; Fig. 5A). To test the specificity of CAMPATH-1H, the same assay was performed in the absence of human complement. This assay demonstrated a dose-(Fig. 5B) and time-dependent (Fig. 5C) reduction of viable MNNG/HOS cells compared to untreated control cells. To determine whether CAMPATH-1H induces apoptosis, we performed Western blot analysis for cleaved PARP. Apoptosis leads to cleavage of the 116 kDa native PARP protein to an 85 kDa fragment. In MNNG/HOS CAMPATH-1H treated cells, no cleaved PARP product was observed, suggesting an apoptosisindependent mechanism of cell growth inhibition (Fig. 5D). antibodies that recognises CD52. The CD52 antigen is highly expressed on lymphocytes, monocytes, eosinophils and in the male reproductive tract 3 . The function of CD52 is still unknown. Nevertheless, the CAMPATH-1H antibody is effective in the treatment of chronic lymphocytic leukaemia [START_REF] Osterborg | Humanized CD52 monoclonal antibody Campath-1H as first-line treatment in chronic lymphocytic leukaemia[END_REF] . Our current work shows for the first time that CD52 is also expressed in non-neoplastic and neoplastic bone-and cartilage-related mesenchymal cells, suggesting a role in bone or cartilage development and progression.

We found high CD52 levels in malignant tumours like chondrosarcoma and osteosarcoma but lower expression in benign and semimalignant tumours. Interestingly, the cartilage-forming tumours showed the highest expression of CD52. A high level of CD52 protein was detected in non-neoplastic cartilage and the benign tumours osteochondroma and chondroma, and an even stronger staining was found in chondrosarcoma. Based on these observations we suggest that CD52 expression in bone tumours correlates with the degree of malignancy. However, due to the small sample number, no statistical correlation between protein intensity and tumour grade could be shown. This observed upregulation of CD52 expression is consistent with reported increased levels of soluble CD52 in patients with chronic lymphatic leukaemia [START_REF] Albitar | Free circulating soluble CD52 as a tumour marker in chronic lymphocytic leukemia and its implication in therapy with anti-CD52 antibodies[END_REF] .

CD52 was also strongly expressed in foetal reactive bone, while quiescent adult bone cells showed weak or no expression of the CD52 antigen. High expression of CD52 in osteosarcoma suggests the reactivation of an embryonic program in these tumours. One of the best known examples indicating the reactivation of embryonic genes in tumours is the expression of the carcinoembryonic antigen (CEA). CD52 and CEA share several properties; for example, patients with osteosarcoma show an increased serum level of CEA [START_REF] Cortes | Carcinoembryonic antigen in osteosarcoma[END_REF] . Comparable to CEA, soluble CD52 was also found in shedding of CD52 might also occur in osteosarcoma cells [START_REF] Albitar | Free circulating soluble CD52 as a tumour marker in chronic lymphocytic leukemia and its implication in therapy with anti-CD52 antibodies[END_REF][START_REF] Giles | Circulating CD20 and CD52 in patients with non-Hodgkin's lymphoma or Hodgkin's disease[END_REF] . These observations suggest that soluble CD52 may act as a tumour marker for chronic lymphatic leukaemia, because increased CD52 levels correlate with aggressive stages of the disease. Whether CD52 can potentially also be used as a tumour marker in the serum of patients with osteosarcoma has yet to be determined.

Microarray analysis of osteosarcoma tissue suggested a significantly higher CD52 expression in lung metastases of osteosarcomas as compared to conventional osteosarcomas. In contrast, the microarray analysis of primary and recurrent giant cell tumours of the bone showed a significantly higher expression of CD52 in primary tumours as compared to the recurrent [START_REF] Guenther | Giant cell tumours of the bone: molecular profiling and expression analysis of Ephrin A1 receptor, Claudin 7, CD52, FGFR3 and AMFR[END_REF] . These differences could not be verified using immunohistochemistry. The differential expression of CD52 was based on variable fractions of lymphocytes and bone tumour cells. For example, in the osteosarcoma metastases, more monocytes and macrophages could be detected than in the conventional osteosarcomas (data not shown) leading to an artificially increased CD52 mRNA level. In the giant cell tumour recurrent tissue, decreased macrophages and lymphocytes were detected as compared to the primary tumours, leading to an artificial decrease in CD52 expression.

Interestingly, an intracellular expression of CD52 flow cytometry could be detected. In a previous study, the GPI-anchored protein EFNA1 was also shown to be expressed intracellularly, which could be due to abnormalities in GPI-anchor biosynthesis or failure of GPI transport resulting in an intracellular localisation of

  investigates levels of CD52 expression in skeletal tumours, especially in giant cell tumours, osteosarcoma and chondrosarcoma tissue and cell lines and their equivalent non-tumorous counterparts. The results imply a role for CD52 in the development and the progression of bone tumours. Furthermore, results obtained in osteosarcoma cells show that CAMPATH-1H leads to a reduction of viable cells.Therefore, CAMPATH-1H might have therapeutic potential for osteosarcoma patients with poor clinical prognosis.

  Tumour tissue samples were obtained from the Institute of Pathology, Charitè Universitätsmedizin Berlin, Berlin, Germany and the Department of Pathology, University of Cologne, Cologne, Germany. Patient characteristics are described in

  : 1. A score to measure the percentage of positively stained cells (PP) was performed as follows: 0 = no staining, 1 = 1-25%, 2 = 26-50%, 3 = 51-75%, 4 = 76-100%. 2. A staining intensity score (SI) was established to measure the intensity of positively stained cells: 0 = no expression, 1 = weak expression, 2 = moderate expression, 3 = strong expression. The combined immunoreactivity score (IRS) value was calculated through PPxSI with a maximum value of 12. Statistical analyses were performed executing the Mann-Whitney-U-Test using SPSS 13.0 software. Differences were considered significant at p<0.05.

  Cells and cell culturePrimary cultures of osteoblasts (HOBc) and chondrocytes were purchased from Provitro GmbH, Berlin, Germany and cultured in special osteoblast growth medium (PromoCell, Heidelberg, Germany) or RPMI1640 media (Biochrom) supplemented with 10% foetal calf serum and 1% penicillin/streptomycine, respectively. The cell line Hut78 was obtained from the ATCC (American Type Culture Collection, United Kingdom) and cultured in RPMI1640 media (Biochrom) supplemented with 10% foetal calf serum and 1% penicillin/streptomycine. The osteosarcoma cell line MNNG/HOS was a gift from Prof. Dr. G. Gaedicke, Clinic for General Paediatrics and Experimental Oncology Charitè Universitätsmedizin Berlin, Berlin, Germany. The cells were maintained in RPMI1640 media (Biochrom) supplemented with 10% foetal calf serum and 1% penicillin/streptomycine. The chondrosarcoma cell line AD was a gift from Dr. T.

( 1 :

 1 10 Serotec) and FITC-conjugated IgG (1:50 Pharmingen) were used for flow , HOBc, MNNG/HOS, chondrocytes, AD and giant cell tumour cell lines. The flow cytometry was performed by staining the cells with the FITC-conjugated antibody in PBS containing 1% BSA (bovine serum albumine) and 0.2% Saponin for intracellular staining and in PBS containing 1% BSA for membranous staining for 30 min. Prior to the intracellular staining the cells were treated with 8% paraformaldehyde for 15 min at 37°C for fixation. Cells were analysed using a FACScan (BectonDickinson).

  did not express CD52 protein, consistent with our Affymetrix mRNA data (Fig.

  no statistically significant correlation between protein intensity and tumour grade could be shown.

  , also known as Alemtuzumab, belongs to the CAMPATH-family of

  patients with chronic lymphatic leukaemia, indicating that a potential

Figure 1 :F:

 1 Figure 1: A: CD52 protein expression in human fetal bone. The osteocytes (blue arrow)

Figure 2 :

 2 Figure 2: Human embryonal cartilage is negative for CD52 (A); also human fetal

Figure 3 :

 3 Figure 3: Chondroma (A), osteochondroma (B), fibrosarcoma (C), aneurysmal bone

Figure 4 :

 4 Figure 4: A-F: Cells were analyzed for surface (mebranous) and cytoplasmic

2 Figure 5 :

 25 Figure 5: Complement-dependent (A), dose-(B) and time-dependent (C) reduction of

Table

  . The tissue specimens were fixed in 4% neutral buffered formaldehyde, and bonecontaining tissue was EDTA decalcified and embedded in paraffin. For each case, hematoxylin and eosin (H&E)-stained slides were carefully reviewed and the diagnosis of the tumour was confirmed according to the WHO criteria by two different pathologists. Tissue microarrays of bone (n=4 foetal and n=6 adult bone samples, each

	6
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	in duplicates; Cat. No. 4011211), cartilage (n=4 embryonal, n=6 foetal and n=10 adult
	cartilage samples, each in duplicates; Cat. No. 4011223) and skeletal tumours (n=3
	fibrosarcomas, n=2 osteosarcomas, n=3 chondrosarcomas, n=1 solitary bone cyst, n=4
	aneurysmal bone cysts, n=1 fibrous dysplasia, n=5 chondromas, n=5 osteomas, n=4
	osteochondromas, each in duplicates; Cat. No. 4132212) were obtained from Provitro
	GmbH, Berlin, Germany. Written informed consent was obtained from each patient.

Table 1 :

 1 CD52. Nevertheless, CAMPATH-1H was able to deplete osteosarcoma cells in vitrowith and without complement. Based on these results, further studies are warranted to Patient samples of tissues used for immunohistochemical analyses.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 :

 2 Immunohistochemical analyses of skeletal tumours. CD52-negative and CD52positive tissue staining and the staining intensity (+: low, ++: medium, +++: strong) was calculated for each sample.

					CD52
	Origin	Tumour	Type	Classification	expression
	Cartilage	Chondroma	Enchondroma Benign	1/1 ++
			Periostal	Benign	3/4 +
		Osteochondroma		Benign	2/3 +
		Chondrosarcoma		Malignant	6/6 +++
	Bone	Osteoma		Benign	5/5 +
		Osteosarcoma		Malignant	14/14 +++
	Fibrous	Fibrosarcoma		Malignant	3/3 +++
	Undefined	Fibrous dysplasia		Benign	1/1 +
		Aneurysmal bone cyst	Benign	3/3 +
	GCT			Semimalignant 26/26 ++

Table 3 :

 3 Membranous and intracellular expression of CD52 analyzed by flow cytometry. %:Percentage of CD52 positive cells. GCT: Giant cell tumour primary culture.

		Membranous		Intracellular
		% IgG	% CD52	% IgG-CD52 % IgG	% CD52	% IgG-CD52
	Hut78	2.0	34.2	32.2	2.3	94.9	92.6
	HOBc	0.3	0.5	0.2	0.3	0.4	0.1
	MNNG/HOS 0.1	9.9	9.8	0.1	69.0	68.9
	Chondrocytes 1.7	2.9	1.2	1.2	8.0	6.8
	AD	2.0	3.3	1.3	0.7	74.4	73.7
	GCT	5.3	25.0	19.7	4.7	54.9	50.2

Published on behalf of the British Division of the International Academy of Pathology Histopathology


P A Marchetti

A Ambrosetti

Z B Su

L Yu

Spin-charge gauge compositeness of electron in HTS: Hints from metal-insulator crossover and entropy behavior

Keywords: numbers: 71.10.Hf, 71.27.+a, 74.25.Fy, 74.25.Gz

We show that the coexistence of Fermi arcs and metal-insulator crossover of the in-plane resistivity can give a hint of a peculiar "gauge compositeness" of the electron in hole-doped high Tc cuprates and a similar hint also comes from the negative intercept at T = 0 of the electronic entropy extrapolated from moderate temperatures in the "pseudogap phase". An implementation of this "compositeness" within the spin-charge gauge approach is outlined and is employed to discuss the above phenomena.

HINTS OF "GAUGE COMPOSITENESS"

We point out two experimental phenomena which suggest, or at least are fully compatible with, a peculiar "compositeness" of the low-energy electron excitation of hole-doped high T c cuprates. This "composite" structure should involve a gapful bosonic constituent and a gapless fermionic constituent, one carrying spin and the other charge, together with a gauge interaction binding them into a resonance. Such a construction is accomplished in particular in the spin-charge gauge approach, developed by us in recent years [1].

The relevant phenomena are:

1) The metal-insulator crossover (MIC) found in the in-plane resistivity decreasing T in heavily underdoped samples, in particular of LSCO and YBCO [2], [3], and in underdoped samples of a variety of cuprates in the presence of a strong magnetic field suppressing superconductivity [4].

2) The negative intercept at T = 0 of the electronic entropy S extrapolated from moderate temperatures in the "pseudogap phase"(PG) [5].

Let us explain why these phenomena suggest a "spincharge gauge compositeness" of the electron.

We start from the MIC. Often this crossover is attributed to disorder-induced localization. However, this interpretation is at odds with the following facts: i) The adiabaticity suggests that MIC with and without magnetic field have the same origin. Including higher doping samples exhibiting MIC, denoting with the mean free path, k F at MIC ranges from 0.1 to 10, i.e. from far below to far above the Ioffe-Regel limit k F ≈ 1, which should characterize a MIC due to disorder localization. ii) In LSCO and YBCO samples with ab inplane anisotropy the MIC temperature of ρ a is different from that of ρ b [6] contradicting the "unique" localization temperature, characteristic of the standard theory of localization. iii) It has been observed a universality of suitably normalized resistivity, ρ n , in terms of T /T * [3] [7], where T * can be identified as the PG temperature. If MIC is intrinsic to the cuprate physics, as follows from the above considerations, it is rather difficult in conventional Fermi-liquid-like theories to reconcile an insulating (semiconducting) behavior of the resistivity with the presence of a Fermi surface (FS), or better Fermi arcs, as detected by ARPES [8]. In fact, the appearance of Fermi arcs suggests the presence of a gapless fermionic excitation, which should produce a metallic in-plane resistivity ρ. The coexistence of FS and insulating behavior of ρ is instead easily accommodated in a slave-particle gauge theory which implies a "gauge compositeness" of the electron. In these approaches, due to the gauge string binding together fermionic and bosonic constituents of the "electron", the velocity of the electron resonance determining the conductivity is dominated by the slowest constituent (Ioffe-Larkin rule [9]). Since the fermionic constituent shows a metallic behavior, exhibiting a FS, the electron resonance has a metallic/insulating behavior if the bosonic constituent does. Furthermore if the dominant contribution is due to the bosonic constituent, it can explain both the insensitivity to details of the FS allowing universality and the negligible effect of non-magnetic impurities affecting only the fermionic constituent (following an original intuition by Anderson [START_REF] Anderson | The Theory of Superconductivity in the High-Tc Cuprates[END_REF]).

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Let us turn to the negative intercept of the extrapolated electronic entropy. A negative intercept suggests a negative contribution to entropy of a "constraint field" reducing the low-energy degrees of freedom. Such a negative contribution naturally arises in a slave-particle gauge approach from the scalar component of the gauge field in two dimensions (in the Coulomb gauge) which does not carry dynamical degrees of freedom [START_REF] Hlubina | [END_REF]. The approximate linear in T behavior of S is consistent with a standard Fermi-liquid contribution and thus can be attributed to the fermionic constituent of the "electron". Ultimately the presence of the gauge field is due to the Gutzwiller projection coming from the strong on-site Coulomb repulsion, hence it should be responsible for the "compositeness".

IMPLEMENTATION IN THE SPIN-CHARGE GAUGE APPROACH

We now outline how the above ideas are actually implemented in the spin-charge gauge approach. In this approach the (approximate) model Hamiltonian for the CuO layers in high T c cuprates is given by the 2D t-J model on a square lattice. The global symmetries of the t-J model corresponding to the SU (2)-spin-rotation and U (1)-charge groups are gauged, i.e. one converting them into local symmetries by coupling the fermion of the model to a spin (SU(2)) and a charge (U( 1))(Chern-Simons) gauge fields, carrying no dynamical degrees of freedom, thus obtaining an equivalent model. A good feature of introducing these gauge fields is that they allow a more flexible treatment of charge and spin responses within a spin-charge decomposition scheme. At the end they will disappear from the game due to a kind of Mean Field treatment, but leaving behind sign of their presence crucial for the low-energy physics, as outlined below. The Gutzwiller projection permits to apply to the fermion of the so obtained gauged t-J model the spin-charge decomposition: One rewrites it as a product of a charged spinless holon and a spin 1/2 neutral spinon (actually via a semionic decomposition [12], [13]). An additional "slave particle" gauge field appears for a correct counting of degrees of freedom. Therefore, treating in "mean-field" the spin and charge gauge fields, the basic dynamical fields of the approach are:

1) Fermionic holons with a finite FS, dressed by spin vortices originating from the spin gauge field, with Fermi momenta k F ∼ δ in PG and ∼ 1δ in the "strangemetal phase"(SM) at higher T or δ, where δ denotes the doping concentration. This change is a remnant of the charge gauge field, which introduces a π flux in PG, thus reducing the Fermi momenta via Hofstädter mechanism. A direct evidence of this small FS might come from a recent experiment on Shubnikov-de Haas oscillations [14].

2) Antiferromagnetic (AF) bosonic spinons with a gap Jm ∼ J δ| ln δ| due to the interaction with the spin vortices, consistent with AF correlation length at small δ derived from neutron experiments [15].

3) Slave-particle gauge fluctuations (in the normal state) dominated by the Reizer singularity [16], due to the finite FS of holons, with an anomalous skin momentum scale Q ∼ (T /t) 1/3 k 2/3 F . For the appearance of Reizer singularity the presence of a gap for spinons is crucial, because gapless spinons would condense at low T thus gapping the gauge field through the Anderson-Higgs mechanism. Gauge fluctuations renormalize holon and spinon lifetimes, renormalizing in particular m to M = (m 2 + ick F T /t) 1/2 in PG. They further bind together spinon-antispinon into a magnon resonance, and holon-spinon into an electron resonance in a momentum slab Q around the FS, with inverse life-time Γ ∼ M in PG, T Q/(tm 2 ) in SM. In PG one has a further reduction of the spectral weight of the electron resonance outside the magnetic Brillouin zone, which bears some resemblance with the appearance of Fermi arcs.

To summarize, in this approach the electron exhibits a "spin-charge gauge compositeness" with the fermionic constituent given by the spinless holon and the bosonic constituent given by the bosonic spin 1/2 spinon.

Let us explain how one recovers the MIC. The in-plane resistivity is calculated via the Ioffe-Larkin addition rule: ρ = ρ s + ρ h , where ρ s is the resistivity of the spinongauge subsystem and ρ h of the holon-gauge subsystem. ρ is dominated by the spinon-gauge scattering and in PG

ρ ∼ ρ s ∼ |M | 1/2 sin(arg M 2 )
.

This formula shows that the spinon gap m combined with the diffusion ∼ T caused by the gauge fluctuations is able to reproduce the MIC. For m 2 >> ck F T /t one finds ρ ∼ T -1 , insulating due to dominating short range AF order. For m 2 < ck F T /t one finds ρ ∼ T α , α ∼ 1/4 metallic, due to dominating diffusion. The above equation implies the universality of the normalized resistivity ρ n = (ρ(T )ρ(T MIC ))/(ρ(T * )ρ(T MIC )) as a function of T /T * ∼ (k F T )/(tm 2 ) . Since in PG k F ∼ δ and m 2 ∼ |δ ln δ|), one obtains a slow decrease of T /T * ∼ T /(|t ln δ|). Here T * can be identified as the temperature of the inflection point in ρ and T MIC of the minimum. The shape of the universal curve derived above agrees semi-quantitatively with the experimental observations [17]. Furthermore the structure of M produces an approximate T 2 behaviour of ρ immediately above the MIC [18], as found experimentally in lightly doped LSCO [19].

Let us turn now to the electronic entropy S. In the spin-charge gauge approach S is the sum of four terms: the contribution of free spinons, S s , of free holons, S h and the fully renormalized contribution of transverse and scalar gauge fluctuations, S ⊥ , S 0 [20]. The spinons are

A c c e p t e d m a n u s c r i p t

massive and thus S s 0 for T lower than the spinon gap (few hundreds K). The holons are described by a Fermi Liquid, therefore the standard result applies: S h ∼ m h T with m h the holon mass ∼ k F , thus S h ∼ δT in PG. S ⊥ and S 0 are estimated using the (approximate) fully renormalized retarded Green function of the gauge field, following [START_REF] Tsvelik | Quantum Field Theory in Condensed Matter Physics[END_REF] and obtaining:

S ⊥ ≈ Q 2 0 ∼ T 2/3 and S 0 ≈ -Λ vF T + 1 v 2 F
T 2 with Λ ∼ J, an UV cutoff. One can see that the scalar contribution contains a negative term, as discussed in general terms above. In this approach the approximately linear behavior of S is basically due to the holons, renormalized by gauge fluctuations. The increase of the slope of S at low δ and its saturation at higher T or δ found experimentally [5] are due to the transition from m h ∼ δ characteristic of PG to m h ∼ 1δ characteristic of SM. The negative intercept is due to the negative scalar gauge contribution and it appears in PG due to the "effective" rather small Fermi temperature, a consequence of the π-flux characteristic of a 2D system, suppressing the positive S ⊥ contribution. The experimentally observed upturn in S/T or γ [5] decreasing T at very low T and above T * are due to the contribution of transverse gauge fluctuations, respectively in PG and in SM. These enhancements of entropy are due to the presence of the gapless transverse gauge mode.

To conclude, we have shown that the "gauge compositeness" of the low-energy electron excitation appearing in the spin-charge gauge approach to the t-J model is able to explain the very unusual phenomena of metalinsulator crossover and negative intercept of electronic entropy, including a number of peculiar experimentally observed features.


S Ejima

S Nishimoto

Ground-state properties of the one-dimensional extended hubbard model at half filling

Keywords: extended Hubbard model, quantum phase transition, bond order wave PACS: 71.10.Fd, 71.10.Pm, 71.30.+h

The density-matrix renormalization group (DMRG) technique is used to study the ground-state properties of the one-dimensional half-filled Hubbard model with onsite (nearest-neighbor) repulsive interaction U (V ) and nearest-neighbor hopping t. We calculate the static spin structure factor to consider the spin degrees of freedom. We notice a striking difference of the static spin structure factor among the spindensity-wave, charge-density-wave (CDW), and bond-order-wave (BOW) phases. Based on the results, we identify the BOW-CDW transition at small (large) U value as continuous (of first order). We also calculate the double occupancy to consider the charge degrees of freedom. For large U , the double occupancy show a discontinuous jump at the BOW-CDW critical point and it implies first order transition. With decreasing U , the jump becomes smaller and vanishes at the tricritical point U t ≈ 5.961t. This value is close to our previous estimation U t = 5.89t obtained with other quantities. Consequently, the results of static spin structure factor and double occupancy support the accuracy of our ground-state phase diagram.

In recent years, the research of quasi-one-dimensional (1D) materials has come once again to the fore in the field of condensed matter physics [START_REF] Giamarchi | Quantum Physics in One Dimension[END_REF]. From the theoretical point of view, one of the simplest models for the materials is the 1D Hubbard model. The ground-state properties of the 1D Hubbard model is well-understood using the exact Bethe-ansatz solution: for any finite on-site repulsion U > 0 at half band-filling, the charge sector is gapped because of the Mott-Hubbard transition and the spin sector is gapless with the dominant 2k Fspin-density-wave (SDW) correlation. It is known, however, that the Coulomb interaction is not sufficiently screened in the 1D materials [START_REF] Egger | [END_REF]3]. And, we also

A c c e p t e d m a n u s c r i p t

add the nearest-neighbor Coulomb interaction V (> 0). Then a rich phase diagram may be expected due to the competition between the effects of U and V : within the g-ology scheme, the system is (basically) insulating with the 2k F -SDW correlation for V U/2 and with the 2k F -charge-density-wave (CDW) correlation for V U/2; furthermore, the existence of a bond-orderwave (BOW) phase between the SDW and CDW phases has been proposed on the basis of non-perturbative numerical results [4]. The system is called as the 1D half-filled extended Hubbard model (EHM), of which Hamiltonian is given by

H = -t i,σ (c † iσ c i+1σ + H.c.) + U i n i↑ n i↓ + V iσσ n iσ n i+1σ , (1)
where c † iσ (c iσ ) is creation (annihilation) operator of an electron with spin σ at site i, and

n iσ = c † iσ c iσ is number operator. t is nearest-neighbor hopping integral and U (V ) is on-site (nearest-neighbor) Coulomb interaction. 0 2 4 6 8 10 U / t 0 1 2 3 4 5 V / t CDW (É c 6 =0; É s 6 =0) BOW (É c 6 =0; É s 6 =0) SDW (É c 6 =0; É s = 0)
Fig. 1. Ground-state phase diagram of the 1D half-filled EHM. The symbols represent the DMRG results as follows: the SDW-BOW phase boundary (filled circles), the continuous BOW-CDW phase boundary (empty squares), the first-order BOW-CDW phase boundary (filled squares), the tricritical point (filled triangle), and the critical end point (empty triangle). The solid and dotted lines correspond to the strong-coupling [7] and the weak-coupling [9] results, respectively.

The ground-state phase diagram of the 1D half-filled EHM is still controversial, though there are a number of analytical [5][6][7][8][9][10] and numerical [11][12][13][14][15] studies. Quite recently, in order to put an end to the controversy, we have reexamined the phase diagram using the density-matrix renormalization group (DMRG) method with considerable accuracy [16]; we determined the SDW-BOW and BOW-CDW phase boundaries based on the results of various physical quantities such as the charge gap, spin gap, Luttinger exponents, and

A c c e p t e d m a n u s c r i p t

BOW order parameter (see Fig. 1). As for the phase boundaries, our results agree quantitatively with the renormalization-group results [9] in the weakcoupling regime (U 2t), with the perturbation results [7] in the strongcoupling regime (U 6t), and with the quantum Monte Carlo results [13] in the intermediate-coupling regime. In addition, we obtained the tricritical point (U t , V t ) = (5.89t, 3.10t) where the BOW-CDW transition changed from continuous to first order, and the critical end point (U c , V c ) = (9.25t, 4.76t) where the BOW phase disappears. In this paper, we calculate the static spin structure factor as well as the double occupancy of the 1D half-filled EHM and show that these results support our previous study [16]. First, we calculate the static spin structure factor S(q) to evaluate the spin degrees of freedom. A drastic change of S(q) could be driven by the phase transitions because the spin structures are quite different among the three phases (see Fig. 1). The static spin structure factor is defined as

0 π/2 π q 0 1 2 3 S(q) : V / t = 1.0 V / t = 2.0 : V / t = 3.0 U / t = 4.0
S(q) = 1 L j,k e iq(j-k) (S z j -S z j )(S z k -S z k ) , (2)
where the sum runs over all sites of the system and • • • denotes the groundstate expectation value. We apply the periodic boundary conditions which are preferred for calculations of the momentum-dependent quantities [17] and study systems up to chain length L = 34. The number of density-matrix eigenstates kept is m = 3000 and the maximum truncation error is 1.0 × 10 -6 . In Fig. 2, we show the calculated results of the static spin structure factor S(q) for U = 4t and L = 34. At U = 4t, the SDW-BOW (BOW-CDW) transition occurs at V ≈ 1.877t (2.164t) [16]. Thus, the three curves in the figure represent results for three different phases; namely, the results for V = t,

A c c e p t e d m a n u s c r i p t

2t, and 3t correspond to the SDW, BOW, and CDW phases, respectively. In the SDW phase, S(q) is basically the same as that of the 1D Heisenberg model [18] and it can be approximately fitted by the 'gross' curve S(q) ≈ ln(1 -q π ). In the BOW phase, one might expect much reduction of S(q) in association with the exponential decay of (S z j -S z j )(S z k -S z k ) with |j -k| driven by the appearance of the spin gap. Indeed, S(q) is just slightly reduced around q = π relative to that in the SDW phase. Since the spin gap is still very small (∆ s ∼ 10 -3 t), the short-range spin-spin correlations must be hardly affected. In the CDW phase, the antiferromagnetic spin-spin correlation seems to be fairly suppressed due to the formation of on-site spin-singlet bound state, though S(q) has still the maximum at q = π. Each the spin-singlet pair is increasingly isolated with increasing V /t and S(q) approaches to zero for all q in the limit of V /U → ∞. Note the zero derivative of S(q) at q = 0 indicates the existence of the spin gap. It is also interesting that the shape of S(q) is similar to that of a gas of bound state, i.e., S(q) ∝ 1cos(q) [19]. Next, we focus on the behavior of S(π) with V to investigate how the antiferromagnetic spin-spin correlation is suppressed. In Fig. 3, we show the system-size dependence of S(π) in the vicinity of the BOW-CDW transition, as a function of V /t with fixed U = 4t and 8t. When U = 4t, we can see gradual decreases of S(π) for all system sizes with increasing V /t, reflecting the rapid development of the spin gap. Although the shape of S(π) becomes steeper as L increases, the transition seems to be continuous in the thermodynamic limit. To confirm it, we scale the inverse slope of S(π) at the critical point with the inverse system size, as shown in the inset of Fig. 3. The extrapolated value to 1/L → 0 is 0.00039. It means that the slope of S(π) at the BOW-CDW transition remains finite in the thermodynamic limit and the transition is continuous. Let us then turn to the case of U = 8t. Differently from the case of U = 4t, S(π) decreases rapidly around the BOW-CDW transition and changes slowly otherwise. The slope of S(π) at the critical point becomes sharper quickly with increasing L and the inverse slope is extrapolated to zero in L → ∞, i.e., the slope diverges

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in the thermodynamic limit. Thus, the BOW-CDW transition for U = 8t must be of first order. The discontinuous transition is concerned with the abrupt charge redistribution from the BOW to CDW phases. We also note that the slow decrease of S(π) below (above) the transition point is associated with very small spin gap (strong charge disproportionation) in the BOW (CDW) phase. Finally, we consider the double occupancy d to confirm that the BOW-CDW transition changes continuous to first order between U = 4t and 8t. We apply the open boundary conditions which allows us to calculate physical quantities quite accurately for very large systems with the DMRG method. We study systems with L ≤ 256 and carry out the finite-size scaling analysis. The number of density-matrix eigenstates kept is m = 2000 and the maximum truncation error is 1.0 × 10 - 9 . With the open boundary conditions, the CDW is a state with a broken translational symmetry and one of two degenerate ground states is picked out by initial conditions of the calculation. Thus, the double occupancy is defined as the average value for central two sites,

d = 1 2 lim L→∞ d L/2 + d L/2+1 , (3)
where d i = n i↑ n i↓ . In the strong-coupling limit U, V t, we find d = 0 in the SDW state and d = 1/2 in the CDW state; in the weak-coupling limit U, V t, we find d = 1/4. If U and V completely cancel each other out, we also find d = 1/4. In Fig. 4, we show the L → ∞ extrapolated results of the double occupancy d as a function of V /t for U = 6t. We can see an increase of d with increasing V /t and a discontinuous jump at the BOW-CDW critical point. The discontinuity clearly indicates that the transition is of first order. We also note that d crosses 1/4 at the critical point. It is consistent

A c c e p t e d m a n u s c r i p t

with the fact that the BOW-CDW transition is derived by the competition between the effects of U and V . Recently, it has been suggested that the double occupancy has no discontinuity for U = 6t using the DMRG algorithm applied to transfer matrices (TMRG) [15]. In the TMRG study, the interval between the calculated points around the critical point is still wider than our estimation of the jump, so that it may possibly include the discontinuity.

Let us now investigate the U-dependence of the discontinuous jump ∆ d . In Fig. In summary, the ground-state properties of the 1D half-filled EHM are studied using the DMRG method. First, we calculate the static spin structure factor to consider the spin degrees of freedom. We notice a striking difference of the static spin structure factor among the three phases with distinct spin structures. Next, we identify the BOW-CDW transition at U = 4t (8t) as continuous (of first order) from the behavior of S(π) near the critical point. Finally, the double occupancy is calculated to consider the charge degrees of freedom. For U = 6t, a discontinuous jump of the double occupancy at the BOW-CDW critical point is found and it implies first order transition. We estimate the tricritical point U t ≈ 5.961t as a point where the discontinuous jump shrinks to zero. This value is quite close to our previous estimation U t = 5.89t obtained with other quantities. Consequently, all the results shown

Fig. 2 .

 2 Fig. 2. Static spin structure factor S ( q) for U = 4t and L = 34. The dotted lines are guides to the eye.

4 UFig. 3 .

 43 Fig. 3. S(π) as a function of V/t at U = 4t (left) and U = 8t (right) for several kinds of chain lengths. The dotted line denotes the BOW-CDW transition point for each U/t values.

Fig. 4 .

 4 Fig. 4. Extrapolated data for the double occupancy d as a function of V/t for U = 6t. The dotted line denotes the BOW-CDW critical point. The dashed line is a guide to the eye.

Fig. 5 .

 5 Fig. 5. Extrapolated data for the jump ∆ d in the double occupancy at the phase transition. The line is a fit ∆ d = 0.139(U -5.961) 0.594 .

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  in this paper support the accuracy of our ground-state phase diagram.

  5, we plot the L → ∞ extrapolated values of the discontinuous jump ∆ d at the BOW-CDW critical point as a function of U/t. For U/t → ∞, doubly occupied sites are alternated with empty sites with no charge fluctuation and, thus, ∆ d is exactly 1/2. With decreasing U/t from U/t → ∞, the charge fluctuation is increasingly allowed and ∆ d decreases. We find ∆ d vanishes just below U/t = 6. Near the vanishing point, the data is well-fitted by a function ∆ d = α(Uβ) γ with α = 0.139, β = 5.961, and γ = 0.594. It leads to the tricritical point U t ≈ 5.961t and it is consistent with our another estimation[16].