CD44 and hyaluronate in the differential diagnosis of dermatofibroma and dermatofibrosarcoma protuberans.

The histological distinction between dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP) may be extremely difficult. CD34 and Factor XIIIa have been used to differentiate DF from DFSP. However, there is an overlap and relative lack of specificity of their expressions. CD44 is a widely distributed integral membrane glycoprotein, which is expressed as a multitude of isoforms generated by alternative splicing of at least 10 different variant exons and post-translational modifications. CD44 is currently thought to be the principal cell surface receptor for hyaluronate (HA), the major component of the extracellular matrix. In this study we aimed to assess the expression of standard CD44 (CD44s) and its isoforms (CD44v3, CD44v4, CD44v5, CD44v6, CD44v7, CD44v7v8, and CD44v10), and HA in DF and DFSP. Immunohistochemical staining was performed on the biopsy specimens of 15 cases of DF and four cases of DFSP, using antibodies that recognize the CD44s, different CD44 isoforms and the hyaluronate binding protein (HABP). Tumor cells displayed a strong CD44s immunoreactivity in all cases of DF whereas a faint HA positivity was observed in the tumor stroma. The DF cells were negative for CD44v3, CD44v4, CD44v6, CD44v7 and CD44v7v8 but showed a strong reactivity for CD44v5 and CD44v10. In contrast, CD44s' expression was significantly reduced or absent in all DFSP lesions and the tumor stroma displayed strong staining for HA. Our results indicate that CD44 and HA can be used as additional diagnostic markers to distinguish DF from DFSP.

Three SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) enhance factor H's cofactor activity enabling MCP-like cellular evasion of complement-mediated attack.

Previously we have shown that two members of the newly named SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins, bone sialoprotein, and osteopontin, bound first to a cell surface receptor and then to complement Factor H thereby blocking the lytic activity of the alternative pathway of complement. Another member of this family, dentin matrix protein 1, is shown in this paper to be very similar to osteopontin in that it can bind strongly to Factor H (K(a) approximately 1 nm) and block the lytic activity through either the vitronectin receptor (alpha(V)beta(3) integrin) or CD44. Binding of Factor H to SIBLING localized to the cells surface was demonstrated by fluorescence-activated cell sorting. Extensive overlapping fragment analyses suggests that both dentin matrix protein 1 and osteopontin interact with cell surface CD44 through their amino termini. Similar fragments of bone sialoprotein, like the intact protein, did not functionally interact with CD44. All three proteins are shown to act in conjunction with Factor I, a serum protease that, when complexed to appropriate cofactors, stops the lytic pathway by digesting the bound C3b in a series of proteolytic steps. These results show that at least three members of this family confer membrane cofactor protein-like activity (MCP or CD46) upon cells expressing RGD-binding integrins or CD44. The required order of the assembly of the complex suggests that this cofactor activity is limited to short diffusional distances.

CD44 in normal human pancreas and pancreatic carcinoma cell lines.

CD44 is an integral cell-surface glycoprotein. Overexpression of the CD44 standard (CD44st) and its variants (CD44v) has been implicated in transformation and progression of many cancer types. Here, we investigated expression of CD44st, CD44v3-7, CD44v7/8, and v10 in five human pancreatic tumor cell lines and normal human pancreatic duct cells transfected with the SV40 large T antigen. CD44st and its variant proteins were quantified using immunocytochemistry and flow cytometry. CD44v7 was expressed at low levels, whereas CD44st, CD44v3, CD44 v4, CD44v, and CD44v6 were expressed at moderate levels in all pancreatic tumor cell lines. In contrast, CD44v7/8 and CD44v10 were expressed at very low levels in two out of the five pancreatic tumor cell lines. Overall, staining of CD44st and CD44 variants was significantly weaker compared to another surface molecule, ICAM-1, reported to be overexpressed in pancreatic cancer cells. Furthermore, the SV40 large T transfected duct cells showed only a weak staining for CD44st, CD44v5, and CD44v6. To determine a possible mechanism for the regulation of surface expression of CD44st, v5 and v6, we incubated Panc-1 cells with bFGF, TGF-beta1, EGF, TNFalpha, and IFNgamma. Only IFNgamma affected the CD44 expression by down-regulation of CD44v6. The constitutive expression of CD44 variants seems to be associated with the malignant state of invasive carcinoma.

Expression of CD44s, CD44v5, CD44v6 and CD44v7-8 in betel quid chewing-associated oral premalignant lesions and squamous cell carcinomas in Taiwan.

Aberrant expression of the cell surface adhesion molecule CD44 and its variant forms has been shown to be associated with the invasive and metastatic potential of cancer cells, and with poor prognosis in several types of cancers. Expression of CD44 standard (CD44s) and variant (CD44v) forms in oral squamous cell carcinoma (SCC), epithelial dysplasia (ED), epithelial hyperkeratosis (EH) and normal buccal mucosa (NBM) have been examined using antibodies to CD44s, CD44v5, CD44v6 and CD44v7-8. Positive CD44s, CD44v5, CD44v6 and CD44v7-8 staining was detected in all the specimens from NBM, EH and ED. Positive staining of CD44s, CD44v5, CD44v6 or CD44v7-8 was detected in 55 (88.7%), 48 (77.4%), 59 (95.2%) and 22 (35.5%) of the 62 SCC specimens, respectively. The positive staining of CD44v7-8 in oral SCC was significantly less than that in NBM (P<0.01). No significant correlation was found between CD44v7-8 expression and daily or total consumption of betel quids or cigarettes by the SCC patients. The 5-year survival rate for patients with CD44v7-8-positive tumours was significantly higher than that for the CD44v7-8-negative group (P<0.03). These results indicate that loss of CD44v7-8 expression may be a valuable factor for determining prognosis in oral SCC patients.

Blockade of metastasis formation by CD44-receptor globulin.

CD44 standard as well as variant isoforms have been frequently reported to be involved in the process of metastasis formation. Whereas in the rat system, but also in some human tumours, the variant exon v6 is of importance in the lymphatic spread of carcinomas, in human malignant melanoma CD44s and, possibly, CD44v10 appear to facilitate local invasion and haematogenous spread. This has been tested in the B16F10 murine melanoma model by treating B16F10-bearing C57BL/6 mice either with a CD44s-/ CD44v10-specific antibody, or with receptor globulins (Rg) containing the extracellular part of CD44s or CD44v10 linked to the constant region of the immunoglobulin kappa light chain. Prior characterization of the CD44s and CD44v10 Rg had shown that both Rgs bound to components of the extracellular matrix, CD44s in particular to hyaluronic acid. Immunohistological screening of organ sections from adult C57BL/6 mice revealed additional evidence for both Rgs binding to elements of the extracellular matrix, particularly in bone marrow, intestine and lung. In the absence of any further treatment, the CD44s Rg reduced the number of lung colonies by 70%, while application of the CD44v10 Rg resulted in 60% reduction. CD44-specific antibodies were equally efficient with regard to B16F10 settlement in the lung. However, only the CD44 Rgs prevented spread and settlement of melanoma cells in distant organs. The finding confirms the involvement of both CD44s and CD44v10 in melanoma progression, and is suggestive for the use of Rgs as therapeutic reagents.

CD44 isoform expression in periodontal tissues: cell-type specific regulation of alternative splicing.

CD44 functions as a receptor for various extracellular matrices and plays crucial roles in homotypic and heterotypic cell-cell interactions. Recently, the molecular structure of CD44 has been extensively analyzed and multiple isoforms produced by alternative splicing of messenger RNA have been identified. In this study, we examined the expression of CD44 isoforms on different cell types isolated from periodontal tissue. In order to examine tissue differences in CD44 isoform expression, we established in vitro cell culture of human gingival fibroblasts (HGF), human periodontal ligament cells (HPDL) and human gingival epithelial cells (HGEC). These cells all expressed CD44 protein and messenger RNA. However, immunoprecipitation and Northern blot analysis revealed that HGEC expressed larger CD44 isoforms than HGF and HPDL. Reverse transcription-polymerase chain reaction with primers flanking the insertion site of alternatively spliced exons was used to study details of the heterogeneity. All cells examined expressed a major band in the absence of alternatively spliced exons and additional larger bands. In particular, HGEC contained more abundant high molecular mass species. In vitro stimulation by IL-1 beta, TNF alpha or phorbol 12-myristate 13-acetate induced an increase in total CD44 messenger RNA in HGF but not change in overall patterns of CD44 isoform expression. However, the isoform expression of HGEC was sensitive to cell density. The amount of larger isoform was decreased by culturing cells beyond confluence. These findings suggest that CD44 isoform expression is cell type-specifically regulated in periodontium and altered according to growth phase of HGEC.

Unaltered strong immunohistochemical expression of CD44-v6 and -v5 isoforms during development and progression of oral squamous cell carcinomas.

The present study was designed to immunolocalize CD44-v6 and -v5 isoforms in normal, dysplastic and malignant oral mucosa as well as in primary and metastatic oral squamous cell carcinomas. Routinely formalin-fixed and paraffin-embedded tissues of 100 oral carcinoma patients were immunohistochemically investigated following wet autoclave antigen retrieval. Both CD44-v6 and -v5 epitopes were uniformly strongly expressed on the cell surface of basal and intermedial epithelial cells of normal and dysplastic mucosa and in all primary and metastatic squamous cell carcinomas with the exception of end-differentiated keratinizing cells. Our results strongly suggest that CD44-v6 and -v5 isoform expression is not altered during development and progression of oral carcinomas. Thus, they seem to be irrelevant factors in the prediction of prognosis in this type of cancer.

Prognostic value of immunohistochemically detected CD44 expression in patients with carcinoma of the vulva.

BACKGROUND: Overexpression of alternatively spliced CD44 isoforms has been reported to correlate with poor prognosis in several human malignancies. To the authors' knowledge, there are no studies concerning the prognostic value of CD44 isoform overexpression in patients with squamous cell carcinoma of the vulva. METHODS: Thirty cases of squamous cell carcinoma of the vulva with International Federation of Gynecology and Obstetrics Stage I to III were examined immunohistochemically for overexpression of CD44 isoforms. We used 3 different variant exon sequence-specific murine monoclonal antibodies to CD44 isoforms containing variant exons v5, v6, and v7 to -8, respectively. The correlation of CD44 overexpression with clinical stage, histologic grade, and disease free and overall survival was investigated. RESULTS: CD44 isoforms CD44v5, CD44v6, and CD44v7-8 were detected in 83% (25/30), 63% (19/30), and 27% (8/30) of the tumor samples, respectively. Patients with tumors overexpressing CD44v6 showed a significantly shorter relapse free (log rank test, P = 0.002) and overall survival (log rank test, P = 0.003) compared with patients with tumors lacking CD44v6 overexpression. Expression of CD44v5 and CD44v7 to 8 had no impact on patients' survival. Clinical stage and histologic grade did not correlate with CD44 overexpression. CONCLUSIONS: Immunohistochemically detected overexpression of CD44 isoforms containing variant exon v6 is correlated with a poor relapse free and overall survival in patients with carcinoma of the vulva.

Expression of the CD44 variant isoforms 6 and 4/5 in breast cancer. Correlation with established prognostic parameters.

Eighty one invasive breast cancers were analysed immunohistochemically to detect if they expressed the adhesion molecules CD44 v6 and v4/5, and the results were evaluated using the semiquantitative IR-score. The results were further divided into four groups: negative, weak positive, moderate positive and strong positive. Fifteen benign breast tumors were also analysed. Sixty eight breast cancers were CD44v6 and v4/5 positive. T3 and T4 cancers showed statistically significant higher positive CD44 rates than T1 and T2 cancers (P < 0.05). We also found a statistically significant correlation between the estrogen receptor and the CD44 status and between the CD44 status and the cathepsin-D status, whereas no correlation between CD44 and the lymph node status, the M status, the grading of the tumors, the progesterone receptor and the menopausal status could be found. Eleven benign tumors were CD44v6 and v4/5 positive. We could not establish any correlation between the expression of CD44 and the metastasizing capacity of breast cancer.

[Expression of CD44 isoforms--a new histopathologic parameter in colorectal carcinoma?]. / Expression von CD44 Isoformen-ein neuer histopathologischer Parameter beim kolorektalen Karzinom?

Expression of CD44H and CD44-isoforms were examined by immunohistochemistry in 102 patients with colorectal tumors in correlation to histological grading, staging and clinical outcome. From normal mucosa to colorectal tumors we found an upregulation of CD44H and CD44-v9 and a de-novo expression of CD44-v6. When compared to histological grading it was found that CD44-v6 expression indicates more differentiated tumors in contrast to less differentiated colorectal carcinomas showing decreasing CD44-v6 expression. Further we came to the result that increasing CD44-v6 expression correlates with progressive tumor stage. In the metastases we found a significant decrease in expression of CD44H, CD44-v9 and-v6 when compared to corresponding primary colorectal tumors. Wether the differential CD44 expression can be used as a prognostic histopathological marker for the progression of colorectal cancer has to be further validated. For final interpretation, our prospective study has to be continued further.

Involvement of CD44 variant isoforms in hyaluronate adhesion by human activated T cells.

The standard, 85-95-kDa form of the hyaluronic acid (HA) receptor CD44 and a number of CD44 mRNA splice variants play important roles in immune responses and tumor metastasis. Variants carrying exon 6 (v6), or 9 (v9) products are transiently expressed on activated human T cells. Here, modulation experiments with specific monoclonal antibodies (mAb) indicate that v6 and v9 are expressed independently on distinct sets of CD44 molecules, and that their combined expression is necessary for HA adhesion. Moreover, the finding that mAb-mediated cross-linking of v6 and v9 promoted cytosolic free Ca2+ mobilization and co-stimulated CD3-triggered T cell proliferation indicates that v6 and v9 possess signaling and effector function activation ability. Finally, HA-mediated signaling appears to be required for variant-dependent adhesion to HA. The observation that soluble HA promoted cytosolic free Ca2+ mobilization indicates that HA-induced Ca2+ mobilization can occur during T cell-HA interaction. Since Ca2+ mobilization was inhibited by pretreatment of cells with an anti-CD44 mAb directed against the HA-binding domain of CD44, CD44 receptors appear to be involved in HA-mediated signal transduction. The requirement of cytosolic free Ca2+ for adhesion is shown by the fact that ionomycin (a Ca2+ ionophore) stimulated, and EGTA (a Ca2+ chelator), inhibited HA adhesion. In addition, cytoskeletal functional activation is required for cell adhesion to HA, since drugs that block actin polymerization, such as cytochalasin B, or actomyosin contraction, such as the calmodulin antagonist W-7, inhibited cell adhesion to HA. As this adhesion is also ADP ribosylation-sensitive, it may involve a GTP-dependent function of CD44v, i.e. ankyrin binding. Our data indicate that there is a functional hierarchy among the CD44 molecules expressed on human peripheral blood T cells and that the splice variants, as compared to the standard form, exhibit a greater HA binding ability which involves CD44-mediated signaling and effector function activation.

Differentiation-associated changes in CD44 isoform expression during normal hematopoiesis and their alteration in chronic myeloid leukemia.

CD44 is a widely expressed, multifunctional, cell-surface glycoprotein that has been implicated in the regulation of normal hematopoiesis. In addition, expression of particular isoforms of CD44 has been associated with malignant transformation and/or the acquisition of metastatic potential. In this study, we used two recently developed monoclonal anti-CD44 antibodies, one reactive with an epitope shared by many CD44 isoforms and the other with an epitope unique to CD44 isoforms containing amino acids encoded by the alternatively spliced exon v10, to compare the expression of CD44 on primitive hematopoietic cells from the marrow of normal individuals and their neoplastic counterparts present in the peripheral blood of patients with chronic myeloid leukemia (CML). Multiparameter fluorescence-activated cell sorter (FACS) analysis and cell sorting studies showed that CD44 is normally expressed at high to very high levels on both long-term culture-initiating cells (LTC-IC) and granulopoietic colony-forming cells (granulocyte-macrophage colony-forming units [CFU-GM]). In contrast, primitive erythropoietic progenitors (burst-forming units-erythroid [BFU-E]) in normal marrow were more homogeneous in their expression of CD44, and very few (less than 5%) showed the very high levels of CD44 seen on 20% to 25% of LTC-IC and CFU-GM. Antibody staining showed the expression of exon v10-containing CD44 isoforms to be restricted to a small subpopulation (4% to 8%) of morphologically recognizable mature (CD34-) myeloid cells within the light-density fraction of normal marrow cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the presence of two exon v10-containing mRNA species. In CML, a significantly greater proportion of the circulating neoplastic CFU-GM expressed very high levels of CD44, and these CFU-GM were accompanied by an increased number of light density v10+ cells, including some that coexpressed CD34. Nonmalignant hematopoietic progenitors mobilized by prior chemotherapy and growth factor treatment of patients with Hodgkin's disease or acute myeloid leukemia in remission showed no changes in CD44 expression relative to normal marrow progenitors. These results provide evidence of early differentiation-associated changes in CD44 expression during normal hematopoiesis in vivo that may be deregulated in the neoplastic clone of patients with CML.

[Immunohistochemical characterization of CD44 molecules expressed in human brain metastases].

CD44v adhesion molecules have been known to be related with carcinogenesis and metastasis of some human cancers including brain metastases of various origin. In this study, we demonstrated further that CD44v expression in brain metastases is extremely heterogeneous. Generally, v5/v6 is homogeneously expressed in the cell populations, while up-regulated v7-v10 could be usually found in a proportion of tumor cells, especially those close to necrosis, at the tumor border and with strong tendency of invasiveness. Our data thus suggest that heterogeneous CD44v expression may be determined not only by intrinsic nature of the cells but also by the surrounding micro-environment. Expression of multiple isoforms of CD44v may confer metastatic cells certain unique abilities, allowing them to localize, then to form metastatic colony in the brain.