Expression of CD44s, CD44v6, and hyaluronan across the spectrum of normal-hyperplasia-carcinoma in breast.

BACKGROUND: The interaction between transmembrane receptors on epithelial tumor cells and the surrounding extracellular matrix molecules is important in tumor progression and metastasis. This interaction is best exemplified by the relationship of the receptor CD44 and the extracellular matrix component hyaluronan (HA). This study seeks to evaluate the expression and the correlation of CD44s, CD44v6, and HA in normal, hyperplastic, and malignant breast epithelium and stroma. MATERIALS AND METHODS: Archival paraffin-embedded tissue from cases of normal breast tissue (n=10), intraductal hyperplasia without atypia (n=13), ductal carcinoma in situ (DCIS) (n=24), stage I infiltrating ductal carcinoma (n=28), stage II infiltrating ductal carcinoma (n=31), and their corresponding positive lymph nodes were retrieved from the surgical pathology files. Tissue sections were evaluated for the expression of CD44s, CD44v6, and HA in the epithelial and stromal cells by immunohistochemistry. RESULTS: Ductal epithelial cells and myoepithelial cells expressed CD44s in all cases of normal and benign breast tissue. The expression of CD44s in breast epithelium progressively decreased with increasing deviation from normal histology: 83% in DCIS, 46% in stage I ductal carcinoma and 26% in stage II ductal carcinoma. The reverse trend was observed for CD44v6 in ductal epithelium: 0% in normal breast, 15% in intraductal hyperplasia, 100% in DCIS, 82% in stage I infiltrating ductal carcinoma, 94% in stage II carcinoma, and 100% of metastatic carcinoma in the lymph nodes. HA was noted exclusively in the stroma but not in the epithelial cells. HA was faintly expressed in the intralobular stroma of normal breast tissue, confined to a narrow faint band adjacent to intraductal hyperplasia and localized to a broad well-defined band around DCIS. Stromal HA staining was more diffuse and intense in infiltrating carcinomas and was particularly pronounced surrounding the metastatic deposits in lymph nodes. CONCLUSIONS: This study demonstrates decreased expression of CD44s accompanied by increased expression of CD44v6 and increased stromal HA in breast cancer. These findings suggest that CD44s, CD44v6, and HA play complementary roles in the development and progression of breast cancer.     

Diagnostic utility of CD44 standard, CD44v6, and CD44v3-10 expression in adenocarcinomas presenting in serous fluids.

CD44 is an 85 to 90 kd integral transmembrane protein encoded by a single 20-exon gene located on the short arm of chromosome 11. In the standard form (CD44s), 10 of the 20 exons are transcribed. Multiple variant isoforms exist (CD44v1-10) which arise from alternate mRNA splicing of the remaining 10 exons. In contrast to the standard form of CD44, which is almost ubiquitously expressed, splice variants are highly restricted in their expression in normal or malignant tissues. The purpose of this study was to evaluate the extent to which metastatic adenocarcinomas in effusions express CD44s, CD44v6, and CD44v3-10 and to assess their diagnostic utility in distinguishing reactive mesothelial cells from adenocarcinomas. Archival paraffin-embedded cell blocks of serous fluids from 23 cases of benign effusions containing reactive mesothelial cells and 45 cases of malignant effusions with metastatic adenocarcinoma (18 ovarian, 11 pulmonary, 9 gastrointestinal, and 7 breast) were retrieved from the surgical pathology files. The cytopathology of all cases was reviewed to confirm the diagnosis. Immunohistochemistry was performed on all cases using antibodies for CD44s, CD44v6, and CD44v3-10 (Bender MedSystems, CA). Positive staining was defined as distinct linear membrane staining. Strong staining in at least 10% of the tumor cells was required to consider the case positive for the particular marker. In benign effusions mesothelial cells expressed CD44s in 22 cases (96%), CD44v6 in 1 cases (4%) and CD44v3-10 in 0 cases (0%). In contrast neoplastic cells in malignant effusions expressed CD44s in 11 cases (24%), CD44v6 in 21 cases (47%), and CD44v3-10 in 39 cases (87%). We concluded that CD44s and CD44v3-10 are useful markers that can be applied to cytologic specimens. CD44s immunostaining can be used as a reliable marker to identify reactive mesothelial cells, meanwhile CD44v3-10 immunostaining can detect majority of adenocarcinomas in malignant effusions.     

Correlation of cytologic examination with ELISA assays for hyaluronan and soluble CD44v6 levels in evaluation of effusions

Hyaluronan (HA) and its major cell surface receptor, CD44, play an important role in tumor growth, proliferation, neovascularization, and invasion. CD44 is an integral transmembrane protein and exists in standard form (CD44s), as well as a myriad of CD44 variants isoforms (CD44v1-v10). Functional fragments of the CD44 can be released from the cell membrane by proteolytic cleavage of extracellular domain producing soluble CD44. Although studies have proposed the use of serum HA and soluble CD44, specifically soluble CD44v6 (sCD44v6) levels, as a tumor markers, its diagnostic utility in body fluid samples has not been clearly established. The purpose of this study was to correlate HA and sCD44v6 levels in effusions with the cytology diagnosis and to assess their usefulness in differentiating between malignant and nonmalignant effusions. In this retrospective study we evaluated HA and sCD44v6 contents in 20 effusions from cytologically positive samples and 10 effusions from cytologically negative samples. Corresponding cytopathology slides were reviewed to confirm the diagnoses. Malignant effusions included 18 cases of metastatic adenocarcinomas (9 ovarian, 3 breast, 3 pulmonary, 3 adenocarcinoma of unknown primary) and 2 cases of lymphomas. The level of HA and sCD44v6 were measured using a sandwich enzyme-linked immunoadsorbent assay. For HA, we used hyaluronic acid quantitative test kit (Corgenix, Denver, CO) and for sCD44v6 we used Human sCD44v6 Instant ELISA (Bender MedSystems, Vienna, Austria). HA concentrations (microg/mL) and sCD44v6 concentrations (ng/mL) were calculated and correlated with clinical data as well as cytodiagnosis. The mean concentration of HA (22.42 +/- 5 microg/mL) and sCD44v6 (70 +/- 42 ng/mL) in the cytologically positive samples was significantly higher than those in the cytologically negative samples for HA (5.5 +/- 5 microg/mL, P < 0.01) and sCD44V6 (17 +/- 10 ng/mL, P < 0.01). Using benign effusions as control and the upper limits of its mean levels for HA (10.5 microg/mL) as the positive boundary value, HA levels exceeded the boundary line in 17 out of 20 malignant effusions and 2 out of 10 benign effusions. Meanwhile, sCD44v6 exceeded the boundary line (27 ng/mL) in 18 out of 20 malignant effusions and 3 out of 10 benign effusions. The calculated sensitivity and specificity of this assay to the diagnosis of malignant effusions were 85 and 80% for HA and 90 and 70% for CD44v6, respectively. We conclude that the HA and sCD44v6 levels in body fluids correlate with the cytology diagnosis and could be used as an ancillary study in cytology to differentiate nonmalignant from malignant effusions.     

Differential expression of CD44s and CD44v3-10 in adenocarcinoma cells and reactive mesothelial cells in effusions

The detection of malignant cells in serous effusions obtained from patients diagnosed with cancer marks the presence of metastatic disease and is associated with a poor outcome. The purpose of this study was to evaluate the role of CD44s and CD44v isoforms in the distinction between mesothelial cells and malignant epithelial cells in effusions. Fifty-nine fresh pleural and peritoneal effusions were studied. These consisted of 41 specimens from patients with known gynecological neoplasms, 9 from patients diagnosed with breast adenocarcinoma, and 9 effusions from patients with various nongynecological malignancies or tumors of unknown origin. Forty-three effusions contained malignant/atypical epithelial cells, and 16 effusions were diagnosed as reactive. Three effusions contained exclusively malignant cells. Specimens were stained with anti-CD44s, v3, v5, v6, v7 and v3-10. The presence of staining in cancer cells, benign mesothelial cells and lymphocytes was evaluated. CD44s immunoreactivity was seen in 10 of 43 (23%) cases in malignant/atypical epithelial cells and in 53 of 56 (94%) cases in benign cells. In contrast, CD44v3-10 was seen in 23 of 43 (55%) cases in malignant/atypical epithelial cells and in 3 of 56 (6%) cases in benign cells. We advocate the use of CD44s and CD44v3-10 immunostaining in diagnostic evaluation of difficult serous effusions. Received: 30 August 1999 / Accepted: 8 November 1999     

CD44s is useful in the differentiation of benign and malignant papillary lesions of the breast

BACKGROUND/AIMS: CD44s, the standard form of CD44, has been shown to be downregulated during malignant transformation of breast cancers. It has also been reported recently to be a useful marker in differentiating between benign and malignant papillary lesions of the breast, with high expression in the former. CD44s expression in benign and malignant papillary lesions was evaluated. METHODS: CD44s expression was assessed by immunohistochemistry in 101 benign papillomas and 59 papillary carcinomas (seven invasive papillary carcinomas, 41 papillary ductal carcinomas in situ, and 11 ductal carcinomas involving papillomas). RESULTS: Patients' age and tumour size were significantly different between the papilloma and papillary carcinoma groups (p < 0.0001). CD44s showed positive staining in 45 papillomas (45%) and five papillary carcinomas (8%), and the difference was significant (p < 0.0001). The myoepithelial cells, when present, were also positive for CD44s in both groups, with no observable differences. Using CD44s positive staining to differentiate between benign and malignant papillary lesions gives a sensitivity, specificity, and accuracy of 45%, 92%, and 62%, respectively. CONCLUSIONS: CD44s may be useful as an adjunct in the evaluation of morphologically problematic cases of papillary lesion of the breast.     

CD44s and CD44v6 expression in localized T1-T2 conventional renal cell carcinomas

To assess the prognostic value of CD44s and CD44v6 tumour expression for patients with T1-T2 conventional renal cell carcinomas, a retrospective immunohistochemical analysis of 95 patients was undertaken. These patients had undergone a radical nephrectomy, performed in three institutions in France between 1987 and 1993. The mean age of the patients was 62.9+/-10.2 years (range from 37 to 85 years) with 66.3% males. At the time of surgery, 84 patients had a T1 and 11 a T2 renal tumour. Fuhrman nuclear grading showed 44 (46.3%) tumours of grade 1, 39 (41.1%) of grade 2, and 12 (12.6%) of grade 3. The mean follow-up period was 58.1+/-36.1 months. At the end of follow-up, eight patients (8.4%) had metastatic disease and no local recurrence was seen. Immunohistochemistry showed that 26 tumours (27.4%) expressed CD44s, but none expressed CD44v6. Statistical analysis showed that CD44s expression was correlated with tumour size (p=0.006) and Fuhrman grading (p<10(-4)). Among the various parameters tested for the multivariate analysis, CD44s expression correlated only with disease-free survival (p=0.04). It is concluded that CD44s expression, but not CD44v6, is of potential prognostic interest in patients with localized T1-T2 conventional renal cell carcinomas.     

CD44 expression in benign and malignant nevomelanocytic lesions

CD44 is an integral membrane glycoprotein that is a principal receptor for hyaluronan and plays a role in cell-extracellular matrix interactions. Recent studies of melanomas in mouse models have suggested that increased CD44 expression by these tumors may relate to metastatic potential. Immunohistochemical expression of CD44 (standard [s] and variant [v6]) in benign and malignant nevomelanocytic lesions was assessed in formalin-fixed, paraffin-embedded tissue and was correlated with histological parameters and prognostic factors. Cases included benign nevi (three junctional, four compound, five intradermal, five blue, six Spitz, one deep penetrating), architecturally disordered (dysplastic) nevi (three, and primary (22) and metastatic melanomas (eight). All of the benign lesions showed diffuse and essentially uniform membrane staining of CD44s in nevomelanocytic cells, regardless of lesion size, depth, or extent of dermal involvement. In contrast, semiquantitative analysis (0 to 3+) of the primary melanomas showed heterogeneous and decreased staining of CD44s, which inversely correlated with lesion size (−0.569) and depth of invasion (−0.622 and −0.617 for Breslow's depth and Clark's level, respectively). These results were significant at P < .05. CD44s expression in metastases paralleled that of their respective primaries. None of the benign nevomelanocytic lesions showed CD44v6 staining. In contrast, all of the malignant nevomelanocytic lesions showed cytoplasmic staining of the tumor cells. Pretreatment with chondroitinase did not alter CD44s staining. CD44s expression by immunohistochemical determination is uniform in benign nevomelanocytic lesions. Malignant melanomas show decreased, heterogeneous staining that inversely correlates with increasing size, depth, and level of invasion. CD44 expression may be a prognostic indicator in malignant melanomas. Tumor staining with anti-chondroitin sulfate monoclonal antibodies suggests that CD44s may be expressed as a chondroitin sulfate proteoglycan in primary melanomas.     

Clinicopathologic significance of expression of CD44s and CD44v6 isoforms in squamous cell carcinoma of the supraglottic larynx

CD44 splice variants are assumed to have a critical role in the malignant progression of many human tumors. However, the clinical significance of CD44 expression is not yet understood. The aim of this study was to investigate the prognostic significance of expression of CD44s and CD44v6 isoforms in squamous cell carcinomas of the supraglottic larynx. CD44s and CD44v6 expression was determined by immunohistochemical analysis of paraffin-embedded tissue specimens from 101 patients. There was a significant correlation between decreased CD44s or CD44v6 expression and a poorer histologic differentiation. No relationship was observed with T stage or nodal metastasis. Decreased CD44s expression, but not CD44v6 expression, correlated with increased recurrence rates. There was no correlation between the decreased expression of any isoform tested and survival. These data confirm a reduction of CD44s and CD44v6 expression in poorly differentiated tumors. However, these changes do not offer a useful adjunct to current prognostic indicators.     

Expression of hyaluronic acid and its receptors, CD44s and CD44v6, in normal, hyperplastic, and neoplastic endometrium

The interaction between epithelial tumor cells and their surrounding stroma is important in tumor progression and metastasis. This is accomplished through a number of transmembrane receptors that interact with stromal extracellular matrix molecules. One of these receptors, CD44, binds to extracellular matrix component hyaluronic acid (HA). The purpose of this study was to evaluate the significance of HA, CD44s, and CD44v6 in benign, hyperplastic, atypical, and malignant endometrial epithelia. Archival paraffin-embedded cell blocks from proliferative endometrium (n = 11), secretory endometrium (n = 12), simple hyperplasia (n = 13), complex hyperplasia without atypia (n = 9), complex hyperplasia with atypia (n = 17), and adenocarcinoma (n = 21) were stained for HA, CD44s, and CD44v6. HA was detected throughout the normal menstrual cycle but was more intense during the secretory phase. Only during the secretory phase was CD44s expressed in the stromal cells in 11 cases (92%), whereas CD44v6 was detected in glandular epithelium in 9 (75%). CD44s was expressed in the glandular epithelium in 2 (15%) cases of simple hyperplasia, 4 (44%) of complex hyperplasia without atypia, 14 (82%) of complex hyperplasia with atypia, and in 16 (76%) of adenocarcinoma. CD44v6 was expressed in the glandular epithelium in 1 (11%) case of complex hyperplasia without atypia, 17 (100%) cases of complex hyperplasia with atypia, and in 18 (86%) cases of adenocarcinoma, but in none of the cases of simple hyperplasia. The endometrial stromal cells expressed CD44v6 in 1 (8%) case of simple hyperplasia, 6 (67%) of complex hyperplasia without atypia, 8 (47%) of complex hyperplasia with atypia, and in 3 (14%) of adenocarcinoma. We concluded that in the normal menstrual cycle, the timing of peak staining of HA and CD44s in the stroma and the up-regulation of CD44v6 in secretory glands are coincident with the period in which the endometrium is most receptive to embryo implantation. HA is more abundant in the stroma adjacent to the tumor, suggesting that interactions between tumor cells and stromal HA promote tumorigenesis. With progression from hyperplasia and with increasing atypia to adenocarcinoma, levels of stromal HA, glandular CD44v6, and glandular and stromal CD44s all increase. Thus, HA and CD44 are both involved in the development and progression of endometrial cancer.     

Expression of CD44 in Canine Mammary Tumours

CD44 is an important adhesion molecule for hyaluronan, a major component of the extracellular matrix (ECM). In human breast cancer, the interaction of tumour cells with the ECM via CD44 is favoured as a major candidate for tumour progression and metastasis. The present study was designed to investigate immunohistochemically the expression of the standard form of CD44 in normal, hyperplastic and neoplastic canine mammary tissue. CD44 was expressed in normal and hyperplastic mammary tissue predominantly by ductal and alveolar epithelial cells and to a minor extent by myoepithelial cells. Stromal cells and blood vessels displayed low to moderate CD44 expression. In simple and complex adenomas and benign mixed tumours there was significant up-regulation of CD44 expression in alveolar epithelial cells compared with adjacent non-neoplastic mammary tissue. Peripheral epithelial cells of simple and complex adenomas, benign mixed tumours and complex carcinomas expressed significantly more CD44 compared with adjacent non-neoplastic mammary tissue. Peripheral epithelial cells of simple adenomas revealed a significantly higher CD44 expression compared with simple carcinomas. A statistical trend to greater CD44 expression was found in peripheral epithelial cells of complex adenomas, benign mixed tumours and complex carcinomas compared with simple carcinomas. Up-regulation of CD44 therefore appears to be associated with benign or relatively benign biological behaviour of canine mammary tumours.     

Expression of CD44s and CD44v6 in transitional cell carcinomas of the urinary bladder: comparison with tumour grade, proliferative activity and p53 immunoreactivity of tumour cells

Alterations of CD44 glycoproteins have been shown to play an important role in progression of various malignancies, including urothelial cancer. We investigated expression patterns of CD44s and CD44v6 in transitional cell carcinoma (TCC) of the urinary bladder in relation to tumour grade, proliferative activity, and immunoreactivity for p53. The selected markers were detected immunohistochemically in 122 samples of TCC. We found a close relationship between CD44s and CD44v6 expression and tumour grade. The extension of positive staining for CD44s and CD44v6 towards the luminal surface was a predominant feature of differentiated carcinomas (grades 1 and 2), suggesting deranged maturation of cancer cells related to their neoplastic transformation. Heterogeneous expression of CD44s and CD44v6 predominated in poorly differentiated tumours (G3-4). However, areas of squamous differentiation within the high-grade tumours displayed strong immunoreactivity for both CD44s and CD44v6. The proliferative activity and p53 overexpression increased with the dedifferentiation of the tumour. The results of this study are discussed in relation to the significance of CD44 expression in TCC and to the explanation for controversial results reported in previous studies on the relationship between CD44 expression and the biological behaviour of urothelial cells.     

Expression and prognostic value of the CD44 splicing variants v5 and v6 in gastric cancer

In the present study, the expression and prognostic role of the CD44 splicing variants v5 and v6 were immunohistochemically investigated in 418 curatively resected gastric carcinomas. CD44v5 was expressed in 65·3 per cent (n=273) and CD44v6 in 77·0 per cent (n=322) of the tumours. Whereas the expression of CD44v5 was correlated with advanced pT categories, with lymph node involvement, and with the presence of blood and lymphatic vessel invasion, such a correlation could not be found for the variant v6. As shown by univariate analysis, patients with CD44v5-positive tumours had a significantly shorter overall survival than patients with CD44v5-negative tumours (P=0·049). In contrast, expression of CD44v6 had no impact on prognosis (P=0·574). In a multivariate analysis including the prognostic parameters pT category and pN category, as well as blood and lymphatic vessel invasion, the prognostic impact of CD44v5 expression could not, however, be maintained. Although in the present study the expression of CD44v5 was correlated with a more aggressive tumour type, these data suggest that neither CD44v5 nor CD44v6 can predict survival in patients with gastric cancer, nor is their expression a suitable tool for identifying subgroups of patients who may be at higher risk. © 1997 John Wiley & Sons, Ltd.     

CD44s and CD44v6 in diagnosis and prognosis of human bladder cancer

The cell adhesion molecules (CAMs) CD44 standard (CD44s) and its variant 6 (CD44v6) are involved in the progression and invasion of human malignancies. However, discrepancies in the prognostic value of CD44s and CD44v6 expression need to be addressed. Aims: To investigate the expression of CD44s and CD44v6 in bladder carcinomas and relate the results to the established prognostic factors. Materials and Methods: 50 bladder carcinoma specimens, 30 cases with transitional cell carcinoma (TCC: 6 bilharzial and 24 nonbilharzial) and 20 cases with squamous cell carcinoma (SCC: 8 bilharzial and 12 nonbilharzial), were included. Immunohistochemical analysis for CD44s and CD44v6 was carried out using avidin-biotin peroxidase method. Results: The level of both CD44s and CD44v6 in TCC was significantly higher in invasive than in preinvasive tumors and normal urothelium (p < .05). A direct association between the percentage of expression of both markers and the grade of TCC (p < .05) was observed. An inverse correlation between CD44s and SCC was seen, where metaplastic urothelium showed higher expression than invasive carcinomas. No association was observed between the expressions of both CD44s and CD44v6 and bilharzial ova, sex and age of the patient, or size of the tumor. Conclusions: The authors report statistically significant correlation between CD44s and CD44v6 expression and increasing grade and stage of TCC. No such correlation with SCC and with bilharzial cystitis, sex and age of the patient, or size of the tumor was documented.     

上皮型钙黏附蛋白、CD44v6及连接蛋白43在肝细胞癌中的表达及意义

目的研究上皮型钙黏附蛋白（E-cad）、CD44v6和连接蛋白43（Cx43）在人肝细胞癌（HCC）中的表达及其表达与患者性别、年龄和组织学分级的关系。方法采用免疫荧光双标记染色技术结合激光扫描共聚焦显微镜观察E—cad、CD44v6及Cx43在30例正常肝组织,25例肝良性病变和38例HCC中的表达;另在HCC组检测并分析了该3种标记物的表达与性别、年龄和组织学分级的关系。结果E—cad与Cx43均在正常肝组织及肝良性病变组高表达,而在HCC组表达都明显降低,前两组与HCC组问表达强度差异均有统计学意义（FE-cad=879．2,F Cx43=303．7,P〈0．05）。相反,CD44v6在正常肝组织及肝良性病变组表达较低,而在HCC中表达较高,差异有统计学意义（F=2057．2,P〈0．05）。HCC组E—cad和Cx43阳性表达强度在患者不同性别、年龄及肿瘤的组织学分级问的差异均无统计学意义（P〉0．05）;而CD44v6表达与HCC的组织学分级有关,HCC分化差,CD44v6表达高（t=-2．06,P〈0．05）,但不同年龄、性别组HCC的CD44v6阳性表达强度差异无统计学意义（P〉0．05）。HCC组E—cad与Cx43的表达呈正相关,而E—cad、Cx43与CD44v6的表达呈负相关。结论HCC的发生、发展伴随着多种分子表型的改变。E—cad、Cx43的低表达与CD44v6的高表达可能参与HCC的侵袭,尤其是CD44v6的表达还与HCC的组织学分级相关。三者联合检测对判断HCC的诊断和预后有一定价值。