Selective Expression of PNA-Binding Glycoconjugates by Invasive Human Melanomas: A New Marker of Metastatic Potential

Alterations of cell-surface glycoconjugates have been associated with invasiveness and metastatic capacity in a number of experimental and human tumors (bladder and colon cancer). We have recently shown that human melanoma cells from variants selected for high metastatic potential in an animal model bind the lectin peanut agglutinin (PNA), and that human melanoma cell populations enriched for PNA binding cells generate a higher frequency of metastases when xenografted into immune suppressed neonatal rats. We have therefore sought cells binding PNA in biopsied human melanocytic tumors and compared frequencies of PNA binding by cells from benign nevi, early and late primary melanomas, and metastatic melanomas. Sections of conventionally processed tissues were deparaffinised and exposed to biotinylated PNA; PNA fixation was revealed by the avidine/peroxidase/AEC technique. In 51 specimens tested, PNA appears to react electively with invasive tumors, since only one of the 7 early primary melanomas (Clark III) reacted while 13/23 late primary melanomas (Clark III-V), and 4/21 melanoma metastases were reactive. In addition, only 1/17 benign nevi bound PNA. In primary tumors, the reactive cells were exclusively invasive tumors cells in the dermis. PNA reactive material was observed in the cytoplasm and plasma membrane of reactive cells. Hence, alterations in composition and cellular localisation of glycoconjugates detectable by lectin histochemistry in melanoma cells may be markers of metastatic potential that may be applicable on an individual patient basis.     

Chromosomal imbalances in primary and metastatic melanomas revealed by comparative genomic hybridization

Characteristic genetic changes underlying the metastatic progression of malignant melanoma is incompletely understood. The goal of our study was to explore specific chromosomal alterations associated with the aggressive behavior of this neoplasm. Comparative genomic hybridization was performed to screen and compare genomic imbalances present in primary and metastatic melanomas. Sixteen primary and 12 metastatic specimens were analyzed. We found that the pattern of chromosomal aberrations is similar in the two subgroups; however, alterations present only in primary and/or metastatic tumors were also discovered. The mean number of genetic changes was 6.3 (range 1-14) in primary and 7.8 (range 1-16) in metastatic lesions. Frequent losses involved 9p and 10q, whereas gains most often occurred at 1q, 6p, 7q, and 8q. Distinct, high-level amplifications were mapped to 1p12-p21 and 1p22-p31 in both tumor types. Amplification of 4q12-q13.1, 7q21.3-qter and 8q23-qter were detected only in primary tumors. The 20q13-qter amplicon was present in a metastatic tumor. The number of genetic alterations were significantly higher in primary tumors which developed metastases within one year after the surgery compared to tumors without metastasis during this time period. Fluorescence in situ hybridization with centromeric and locus-specific probes was applied to validate CGH results on a subset of tumors. Comparison of FISH and CGH data gave good correlation. The aggressive behavior of melanoma is associated with accumulation of multiple genetic alterations. Chromosome regions, which differ in the primary and metastatic lesions, may represent potential targets to identify metastases-related chromosomal alterations.     

Variation of ER status between primary and metastatic breast cancer and relationship to p53 expression*.

OBJECTIVE: Estrogen-dependent growth of breast cancer can be blocked by anti-estrogens. Estrogen receptor (ER) presence in breast cancer implies responsiveness to endocrine therapy. However, for those patients who ultimately develop resistance to endocrine therapy, the mechanisms remain unclear. The present study attempted to compare the expression status of ER mRNA in a series of primary breast tumors with matched metastases and explored the relation between ER and mutant p53 expression. METHODS: In situ hybridization using a digoxigenin-labeled estrogen receptor cDNA probe was employed to determine the expression of ER mRNA in 52 cases of primary tumors and their matched axillary lymph node metastases. Immunohistochemical staining using a monoclonal antibody against ER was also performed. RESULTS: ER expression was observed in 53.8% (28/52) of primary tumors and 48% (25/52) of metastases, while 57.7% (30/52) of primary tumors and 53.8% (28/52) of metastases showed ER mRNA positivity. There were variations in ER status between in situ hybridization and immunohistochemistry measurements and between primary tumors and metastases. Mutant p53 expression was inversely associated with ER-negative, high-grade tumors. CONCLUSIONS: In situ hybridization may be a more specific and sensitive method for determination of ER status than immunohistochemistry. It is possible that the biologic properties of ER change, and these changes may influence tumor response to endocrine therapy. In view of the ER variation, it was suggested that the ER status of metastatic tumors in addition to primary tumors should be taken into consideration in order to better determine the benefit of clinical endocrine therapy.     

Comparison of nuclear DNA content in primary and metastatic malignant melanoma.

The DNA patterns obtained from 23 primary malignant melanomas and 35 corresponding metastases were compared and found to differ in many cases. In eight cases the primary tumors and their metastases had a ploidy type I ("euploid") DNA pattern. One case had a type I primary tumor and both type I and type II metastases. Five cases had type I primary tumors and ploidy type II ("aneuploid") DNA pattern metastases. In five cases the primary tumors and corresponding metastases were type II, and in another four cases the primary tumors were type II, whereas the metastases were type I. We interpret these data as indicating that malignant melanomas (more often than adenocarcinomas) are composed of genetically heterogeneous tumor sublines that frequently give rise to heterogeneously composed metastases. Since we sometimes observed a change in the DNA content in malignant melanomas, it seems to be more difficult to obtain prognostic information from DNA analysis in malignant melanoma as compared to the more stable adenocarcinomas.     

Immunohistochemical expression of retinoblastoma gene product (Rb), p53 protein, MDM2, c-erbB-2, HLA-DR and proliferation indices in human urinary bladder carcinoma

Archival biopsy specimens from transitional cell bladder cancers (n=88) were analysed immunohistochemically for the expression of the retinoblastoma (Rb) gene protein, p53, mdm2, c-erbB-2, HLA-DR antigen and proliferation indices. An altered nuclear expression of Rb, p53 and mdm2 was observed in 55.2%, 33.3% and 18.2% of tumors respectively. Cytoplasmic membrane immunoreactivity (>25% tumor cells) for c-erbB-2 was detected in 14.1% of tumors and aberrant HLA-DR antigen cytoplasmic staining (>5% of tumor cells) in 22.2% of the cases. P53 overexpression was associated with higher tumor grade and stage. Aberrant HLA-DR antigen expression and PCNA were also correlated with the grade of differentiation and tumor stage. MIB1 was statistically correlated with stage. pRb scores and HLA-DR antigen expression were correlated with proliferation activity as determined by PCNA and MIB1 immunostaining. p53 protein was also strongly correlated with the proliferation index PCNA. A strong correlation between PCNA and MIB1 (p<0.0001) was also found. In addition a statistically positive correlation between p53 and HLA-DR antigen expression was observed. Our data show that, although pRb and p53 protein expressions are not associated between them, they may contribute to the growth fraction of the bladder cancer. In addition, p53 and HLA-DR antigen expression could be indicators of aggressive behavior of bladder cancer.     

Volume-corrected mitotic index and p53 immunoreactivity in borderline and malignant epithelial ovarian tumors

OBJECTIVE: To determine if there are differences in p53 immunoreactivity and proliferation rate using the volume-corrected mitotic (M/V) index between borderline and malignant ovarian tumors. STUDY DESIGN: Thirteen borderline (7 serous and 6 mucinous) and 17 malignant (14 serous and 3 mucinous) ovarian neoplasms were included in this study. M/V index was calculated according to the formula of Haapasalo et al. Formalin-fixed, paraffin-embedded tissue sections were immunostained with a monoclonal antibody against p53 protein using an avidin-streptavidin method. RESULTS: Carcinomas had a considerably higher M/V index (17.7 +/- 9.8) than borderline tumors (1.5 +/- 1.7, P < .001). The usual mitotic index was also higher in carcinomas than in borderline tumors (24.9 +/- 16.6 versus 1.6 +/- 1.9, P < .001). p53 Immunoreactivity was found in 5/17 carcinomas (29.4%), while all borderline tumors were negative. CONCLUSION: Even though the number of our cases was too small to draw definitive conclusions, our results suggest that M/V index and p53 immunoreactivity may be of some help in the differentiation between borderline and malignant ovarian epithelial tumors.     

Expression profile of SIRT2 in human melanoma and implications for sirtuin-based chemotherapy

Melanoma is cancer of melanin-containing melanocyte cells. This neoplasm is one of the most deadly forms of skin cancer, and currently available therapeutic options are insufficient in significantly improve outcomes for many patients. Therefore, novel targets are required to effectively manage this neoplasm. Several sirtuins have previously been found to be upregulated in melanoma, so in this study, the expression profile of SIRT2 was determined. Employing a tissue microarray containing benign nevi, primary melanomas, and lymph node metastases, we have found that the tissue from lymph node metastases appears to have a significant upregulation of SIRT2 relative to primary tumors across the nuclear, cytoplasmic, and whole cell data. Additionally, SIRT2 staining was found to be higher in the nucleus of metastatic melanomas compared to cytoplasmic staining. As SIRT2 is considered to be a predominantly cytoplasmic protein, this is a novel and very interesting finding. This, combined with previous studies that show other sirtuins are increased in melanoma and involved in cellular proliferation and survival, leads to the suggestion that exploring pan-sirtuin inhibitors may be the best target for the next iteration of melanoma chemotherapeutics.     

Fli-1 expression in malignant melanoma

Friend leukemia integration site 1 (Fli-1) has been reported as the first nuclear marker of endothelial differentiation; it is expressed in leukocytes and recently demonstrated in melanomas. Formalin-fixed, paraffin-embedded tissue sections from 97 melanomas including 69 cases of primary and 28 metastatic melanomas were evaluated by immunohistochemistry. Five melanoma cell lines were evaluated by Western blot and immunocytochemistry. Fli-1 expression was observed in all cell lines. Fli-1 expression was higher in metastatic than in primary tumors (r=0.208, p=0.041, Spearman correlation), it positively correlated with Ki-67 expression (r=0.233, p=0.022, Spearman correlation), and the presence of an ulcer in the primary tumor (r=0.267, p=0.030, Spearman correlation). Therefore, the expression of Fli-1 in malignant melanoma appears to be associated with biologically more aggressive tumors.     

Immunophenotyping of human HT29 colon cancer cell primary tumours and their metastases in severe combined immunodeficient mice

The immunophenotype of HT29 human colon cancer cells implanted into severe combined immunodeficient mice was assessed in primary tumours and their metastases in the lungs using an indirect immunohistochemical method. After primary tumours were surgically removed, the metastases were given time to develop, thus paralleling the clinical situation. While vimentin was negative in both primary and secondary tumours, E-cadherin was present as membrane-bound labelling in the primary tumours only. Whereas the markers p53, MIB1, PCNA and CEA were consistently positive in both primary and metastatic tumours, CD44 variant 6 and CA125 were negative in metastases but positive in the primary tumours. There was a significant increase in the percentage of cells labelled for p53 in the primary tumours compared with the metastases. For the proliferation markers, there was no significant difference in labelling between primary tumours and metastases for MIB1. Of the cytokeratins examined, CK 20 gave the strongest and most consistent reaction in both primary and secondary tumours. The results indicate that, for certain immunohistochemical markers, results are the same in both primary tumours and metastases. Hence, in these cases, antigens that are expressed on the primary tumour as well as on the metastases can serve as target molecules for immunologically based forms of treatment of metastases. This revised version was published online in November 2006 with corrections to the Cover Date.     

Immunophenotyping of human HT29 colon cancer cell primary tumours and their metastases in severe combined immunodeficient mice

The immunophenotype of HT29 human colon cancer cells implanted into severe combined immunodeficient mice was assessed in primary tumours and their metastases in the lungs using an indirect immunohistochemical method. After primary tumours were surgically removed, the metastases were given time to develop, thus paralleling the clinical situation. While vimentin was negative in both primary and secondary tumours, E-cadherin was present as membrane-bound labelling in the primary tumours only. Whereas the markers p53, MIB1, PCNA and CEA were consistently positive in both primary and metastatic tumours, CD44 variant 6 and CA125 were negative in metastases but positive in the primary tumours. There was a significant increase in the percentage of cells labelled for p53 in the primary tumours compared with the metastases. For the proliferation markers, there was no significant difference in labelling between primary tumours and metastases for MIB1. Of the cytokeratins examined, CK 20 gave the strongest and most consistent reaction in both primary and secondary tumours. The results indicate that, for certain immunohistochemical markers, results are the same in both primary tumours and metastases. Hence, in these cases, antigens that are expressed on the primary tumour as well as on the metastases can serve as target molecules for immunologically based forms of treatment of metastases. This revised version was published online in November 2006 with corrections to the Cover Date.     

Alterations of the p53 gene in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignancy which is consistently associated with the Epstein-Barr virus (EBV). The structure of the EBV genome in NPC suggests that NPC is a clonal proliferation of epithelial cells which emerges after EBV infection. The disease develops with high incidence in specific populations in discrete geographic locations, implicating possible genetic or environmental cofactors. Mutations of the p53 gene are among the most frequent genetic changes found in a large variety of human tumors. Mutations in p53 have been shown to abrogate the suppressor function of wild-type p53 and thus contribute to the transformed phenotype. To determine if mutation in p53 participates in the development of the malignant clone in NPC, the structure and sequence of p53 in 42 primary, metastatic, and nude mouse-passaged NPC specimens was analyzed. A high frequency (6 of 9) of mutations was detected in the nude mouse-passaged tumors, while only 2 of 15 metastatic and 0 of the 18 primary tumors harbored mutant p53. The p53 mutations included single-point mutations and more extensive changes such as frame shifts, deletion, duplication, or complete loss of coding sequences. These data indicate that alterations of the p53 gene are unlikely to be involved in the initial genetic events leading to the clonal outgrowth in NPC. However, although it is a rare NPC which can be established in nude mice, this growth advantage appears to be conferred on tumors bearing a mutant p53.     

Detection of proliferating cell nuclear antigen in diagnostic histopathology.

We describe the effects of tissue preservation, fixation time, and hydrolytic treatment on the detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining with three commercial anti-PCNA antibodies (19A2, 19F4, PC10). Our goal was to provide guidelines for PCNA immunohistochemistry in formalin-fixed, paraffin-embedded specimens. In proliferative cell compartments, nuclear staining was achieved with all three antibodies. In some cases PCNA was also expressed in non-proliferative, histologically normal tissues associated with tumors or other lesions elsewhere. In most autopsy specimens PNCA immunoreactivity was markedly diminished as compared with similar surgical specimens. Incubation overnight with primary antibody at 4 degrees C enhanced PCNA immunoreactivity over incubation at 42 degrees C for 45 min. Pre-treatment with 2 N HCl did not increase staining. Staining with the PC10 antibody was much better preserved than staining with the antibodies 19A2 and 19F4 after prolonged formalin fixation of surgical specimens and in tissues obtained at autopsy. With all three antibodies, however, PCNA immunoreactivity was well preserved during formalin fixation for 8-24 hr and during fixation delays for 8 hr at room temperature. This indicates that PCNA is stable under conditions routinely encountered in diagnostic surgical pathology and facilitates its potential use as a diagnostic proliferation marker.     

DNA ploidy pattern and S-phase characteristics of metastatic melanoma

Samples of 130 metastatic melanomas from 92 patients were analyzed by DNA flow cytometry. DNA aneuploidy was observed in 67% of the patients. DNA indices were evenly distributed from 0.6 to 2.6 Tumors originating from primary lesions in the lower extremities were more frequently DNA aneuploid than those of other sites. S-phase fraction (SPF) was evaluable from 73 tumors. DNA aneuploid tumors had a significantly higher SPF than diploid tumors, and females had a higher SPF than males. Furthermore, distant metastases had a higher SPF than metastases in regional lymph nodes and in transit metastases, probably indicating a higher growth potential in metastases spreading to distant sites.     

Immunohistochemical localization and correlation of p53 and PCNA expression in breast carcinoma

The object of the present study is to detect the p53 tumour suppressor gene and proliferation cell nuclear antigen (PCNA) expression in breast carcinoma by immunohistochemistry and correlate them with the prognostic parameters. Total 35 cases of primary breast carcinoma were studied and classified histologically. Paraffin sections were stained by using monoclonal antibody D07 for p53 protein and PC-10 for PCNA. Out of 35 cases, 16 (45.7%) were p53 positive and 25 (71.4%) were PCNA positive. The mean PCNA labelling index (PCNA LI +/- SD) was 58.97 +/- 22.72 in tumors positive for both p53+ and PCNA+ while cases negative for p53- and positive for PCNA+ has higher PCNA LI +/- SD (59.24 +/- 18.97). The difference in the two groups was not significant. Most cases were positive for both p53+ and PCNA+ in the age group < 30 with higher mean PCNA LI +/- SD (62.20 +/- 27.13) than in the group > 30 (57.88 +/- 18.47). In the pre-menopausal group 57.1% cases were positive for p53+ with higher PCNA LI +/- SD (59.94 +/- 24.22). Maximum p53 and PCNA positivity was observed in grade III tumors (63.2% and 84.2%). The mean PCNA LI +/- SD was also highest in grade III carcinomas (66.83 +/- 13.97). No significant correlation was found between p53 and PCNA status with morphological type and tumour size except that logistic regression showed a positive correlation with tumour grade. Therefore the present study suggests that both p53 expression and PCNA are markers of poor differentiation in breast cancer.     

Fine needle aspiration biopsy cytology of paragangliomas. Cytologic, light microscopic and ultrastructural studies of three cases

Paragangliomas are uncommon tumors arising in paraganglion tissue, which is especially well represented in the head and neck. In our hospital three head and neck masses recently proved to be paragangliomas on aspiratory biopsy cytology, with subsequent histologic confirmation. One was an apparently benign carotid body tumor, while the others were metastatic nasopharyngeal paragangliomas. The latter two tumors were clearly malignant, with roentgenographically proven pulmonary metastases in one and widespread lymph node involvement in the other. Histologically all were vascular tumors composed of large cells with abundant cytoplasm and pleomorphic, sometimes bizarre nuclei. These characteristic chief cells were clearly evident on cytology, with pleomorphic nuclei, prominent nucleoli, clumped chromatin and generally eosinophilic, finely granular or reticular cytoplasm. However, the distinction between benign and malignant tumors depended on the presence of mitoses or a tumor diathesis. Cytology seems to be able to provide a diagnosis of paraganglioma and, in these cases at least, an estimation of the biologic behavior.     

The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intradermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient's age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing melanoma from melanocytic nevi, if we consider c-Kit expression in intraepidermal proliferating cells. The c-Kit expression in proliferating melanocytes in the dermis could help in the differential diagnosis between a superficial spreading melanoma (with dermis invasion) and a compound nevus or an intradermal nevus. Finally, c-Kit could be a good diagnostic tool for distinguishing benign compound nevi from malignant melanocytic lesions with dermis invasion and to differentiate metastatic melanoma from primary melanoma.     

Immunohistochemical detection of nestin in pediatric brain tumors.

Nestin is an intermediate filament protein (IFP) expressed in undifferentiated cells during CNS development and in CNS tumors. Previous studies have arrived at different conclusions in terms of which types of CNS tumors express nestin. In this report we establish an immunohistochemical protocol using antigen retrieval, which significantly enhances staining with two polyclonal anti-nestin antisera, #130 and #4350. The staining pattern was identical for the two nestin antisera and very similar to that of vimentin, while glial fibrillary acidic protein (GFAP), immunoreactivity was absent from 9.5-week-old forebrain. The current study of 20 primary CNS tumors from pediatric patients included seven ependymomas, seven primitive neuroectodermal tumors (PNETs), five pilocytic astrocytomas, and one glioblastoma multiforme (GBM). All these tumors expressed nestin to various extents, in contrast to five brain metastases tested. Strong nestin immunoreactivity was found in malignant primary CNS tumors, whereas benign pilocytic astrocytomas showed low but consistent nestin expression. In all tumors nestin immunoreactivity was confined to the cytoplasm of tumor cells and was co-expressed with astrocyte markers vimentin, GFAP, and S-100. Vascular endothelial cells of all neoplasms also showed marked immunoreactivity for nestin and vimentin, whereas they were negative for GFAP and S-100. In conclusion, antiserum #4350 detected nestin in formalin-fixed, paraffin-embedded tissue sections by heat-induced antigen retrieval immunohistochemistry. Nestin was expressed in both highly malignant and low malignant gliomas, indicating the potential use of nestin as a diagnostic tumor marker in surgical pathology.     

DNA ploidy pattern and S-phase characteristics of metastatic melanoma.

Samples of 130 metastatic melanomas from 92 patients were analyzed by DNA flow cytometry. DNA aneuploidy was observed in 67% of the patients. DNA indices were evenly distributed from 0.6 to 2.6 Tumors originating from primary lesions in the lower extremities were more frequently DNA aneuploid than those of other sites. S-phase fraction (SPF) was evaluable from 73 tumors. DNA aneuploid tumors had a significantly higher SPF than diploid tumors, and females had a higher SPF than males. Furthermore, distant metastases had a higher SPF than metastases in regional lymph nodes and in transit metastases, probably indicating a higher growth potential in metastases spreading to distant sites.     

High expression of Wee1 is associated with poor disease-free survival in malignant melanoma: potential for targeted therapy

Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options. The Wee1 kinase is a major regulator of the G(2)/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes. We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21(Cip1/WAF1) (p = 0.003), p53 (p = 0.025). Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008). Transfections using siWee1 in metastatic melanoma cell lines; WM239(WTp53), WM45.1(MUTp53) and LOX(WTp53), further support our hypothesis of a tumor promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G(1)/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents.