Serum CD44 splice variants in cervical cancer patients

Aberrant expression of the cell adhesion molecule CD44 has been detected in human tumours and has been shown to be associated with metastasis and poor prognosis in human malignancies. We evaluated serum levels of different soluble CD44 molecules (CD44 standard form and CD44 splice variants v5 and v6) in cervical cancer patients stage IB to IIIB. Two-hundred three serum samples were analysed. Serum levels of CD44st and CD44v5 showed no significant correlation with the presence or absence of cervical cancer. The splice variant CD44v6 showed a mean concentration of 227.3 ± 90.9 (minimum 71.4, maximum 543.9) ng/ml when tumour was present and a mean concentration of 198.7 ± 135.4 (minimum 67.2, maximum 696.3) ng/ml in cases of complete remission (P-value = 0.0001). However, in this preliminary study the sensitivity/specificity characteristic of CD44v6 was poor.     

Expression of CD44s and CD44 splice variants in human melanoma.

The ability of tumor cells to adhere and detach from extracellular matrix and endothelial cells, is a crucial step in the metastatic process and may alter the clinical prognosis of some human tumors such as melanomas. CD44, the major cell surface receptor for hyaluronate, has been implicated in cell adhesion and in tumor progression. We studied the expression of standard CD44 molecule (CD44s) and its variants v3 and v6 in 57 human primary melanoma biopsies, without previous treatment. We analyzed the association between CD44 expression and the principal clinicopathological features, including survival. Fifty-six of 57 tumors expressed CD44s, associated to the cytoplasmic membrane. No expression of CD44v3 or CD44v6 was detected. No association between CD44s expression and prognostic factors such as tumor thickness, growth type, stage or anatomic site of the lesion was found. However, a positive correlation between CD44s expression and Clark level (Spearman, p<0.001) was found. While only 33.3% of melanomas Clark I + II showed high expression of CD44s (more than 50% of positive cells), 82.6% of melanomas Clark IV + V did so. Kaplan-Meier analysis revelead that patients whose melanomas had high expression of CD44s showed a reduced relapse free survival (RFS) rate, though without statistical significance. No difference between the level of CD44 expression and overall survival (OS) was found. We conclude that melanomas only expressed CD44s, and that its level was associated with Clark's stage. CD44s seems not to be useful as a tumor marker, because it does not predict either RFS or OS.     

Immunohistochemical and serological evaluation of CD44 splice variants in human ovarian cancer.

The surface glycoprotein CD44 is widely distributed in different tissues. In contrast to healthy tissue, tumour samples show a more complex pattern of CD44 expression, indicating a loss of splice control. Beside cell-surface expression, the measurement of soluble CD44 in serum of cancer patients could be useful in early diagnosis and assessment of disease status. We evaluated the surface expression of CD44 isoforms in 22 ovarian cancer patients by means of immunohistochemistry. Additionally, we investigated 134 serological samples of these patients for the occurrence of CD44 isoform expression. For CD44 standard, CD44v5 and CF44v6 mean serum levels in patients with clinically detectable or non-detectable ovarian cancer were 422.4 +/- 143.8 ng ml-1 and 547.4 +/- 148.2 ng ml-1, 12.3 +/- 7.9 ng ml-1 and 21.9 +/- 12.2 ng ml-1 and 105.5 +/- 37.9 ng ml-1 and 144.9 +/- 50.9 ng ml-1 respectively (P-values not significant). CD44 surface proteins containing epitopes encoded by splice variants CD44v5, CD44v6 and CD44v7-8 were immunohistochemically detected in 9% (n = 2), 13% (n = 3) and 4% (n = 1) of the 22 tumour samples respectively. In the present study we showed that in ovarian cancer CD44 isoforms CD44v5 and CD44v6 are expressed in very low amounts by the tumours. In accordance with this, we found that the presence of tumour is not associated with higher serum levels of CD44standard, CD44v5 and CD44v6 in preoperative serum samples in ovarian cancer patients.     

CD44 expression patterns in breast and colon tumors: A pcr-based study of splice variants

CD44 cell-surface receptor expresses multiple isoforms, some of which are believed to play a role in tumor growth and metastasis. The CD44 gene is composed of 19 exons, of which 9 (exons 6 to 14) are alternatively spliced to form inclusions in the intervening membrane proximal region. Sequences present in the shortest metastatic variant cloned from a rat metastatic cell line have been shown to correspond to human exons 10 and 11, also called exons v6 and v7. Using RT-PCR, we have addressed in detail the CD44 isoforms produced in human breast and colon tumors. We analyzed 53 breast-tumor- and 58 colon-tumor-related samples as well as I benign mastopathy, I normal breast, 4 non-invaded lymph nodes and 8 normal colon tissues. All tumors analyzed expressed the hemopoietic CD44 (CD44H) isoform (no alternatively spliced exons added), whereas 81% expressed the CD44E form (addition exons 12, 13 and 14). Furthermore, 85% of tumors presented complex patterns of expression, with an average number of 5 to 6 bands detected. In view of their implication in the metastatic process, we investigated in greater detail the isoforms containing exons 10 and 11 (v6 and v7). Exon 10 was more frequently expressed than exon 11, 80% and 57% of the samples respectively. The great majority of cases showed ladder-like patterns starting from the shortest forms (exons 5-10 or 5-10-11) and larger-molecular-weight bands corresponding predominantly to sequential inclusions of exons from 3' to 5'. Exon-10 and exon-11 variants were also found in one benign mastopathy. The majority of normal tissues (1 breast and 6/8 colon) expressed only the CD44H isoform. These data indicate that expression of metastatic variants is common in human breast and colon tumors and can occur early during cancer progression, as testified by their presence in a benign breast tumor. While expression of exon-10 variants were correlated with presence of distal metastases in colon tumors, exon-11 variants were not (metastatic events were too rare in our breast-tumor series to reach significance). This suggest that exon 10 may correspond to the minimal sequences required to favor metastatic events. © 1995 Wiley-Liss, Inc.     

Schwann cell tumors express characteristic patterns of CD44 splice variants

Summary Members of the CD44 family of cell surface hyaluronate-binding proteins have been implicated in cell migration, cell-matrix interactions and tumor progression. To determine whether these proteins might play a role in the normal functions of Schwann cells and in their tumorigenesis, we examined the patterns of CD44 expression in Schwann cells from rat peripheral nerve, rat Schwann cell tumor lines, and human schwannomas. Normal rat spinal nerves and primary Schwann cell cultures expressed standard CD44 (CD44s) but not alternatively spliced variant isoforms. In contrast, rat Schwann cell tumor lines expressed both CD44s and a number of variants, including proteins containing sequences encoded by exon v6. Furthermore, we found that these cell lines bind hyaluronate, and that their cell surface hyaluronate binding correlates with CD44 expression. All of the human schwannomas also expressed CD44 variants, especially epitopes encoded by exon v5, the border between v7 and v8, and v9-10. These data indicate that Schwann cells normally express CD44s, that Schwann cell tumors express both CD44s and particular variants of CD44, and that CD44s and possibly variants of CD44 are involved in hyaluronate recognition by Schwann cell tumors.     

Immunohistochemical detection of adhesion molecule CD44 splice variants in lymph node metastases of cervical cancer

Expression of specific cell adhesion molecule CD44 isoforms (splice variants) has been shown to be associated with poor prognosis in human cervical cancer. We used 3 different variant exon sequence-specific murine monoclonal antibodies (MAbs) to epitopes encoded by exons v5, v6 and v7-v8 of human variant CD44 to study the expression of CD44 splice variants in 35 primary squamous-cell carcinomas of the cervix and pelvic lymph node metastases by means of immunohistochemistry. Primary tumors showed expression of CD44 splice variants CD44v5, CD44v6 and CD44v7-8 in 93%, 73% and 33% of cases, respectively. Lymph node metastases expressed CD44v5, CD44v6 and CD44v7-8 in 83%, 53% and 21% of cases, respectively. Tumors with expression of CD44v6 in pelvic lymph node metastases showed metastatic spread to 2 or more pelvic lymph nodes significantly more often compared to patients without expression of splice variant CD44v6. Patients suffering from tumors with lymph node metastases expressing splice variant CD44v6 had a poorer recurrence-free survival compared to patients without CD44v6 expression in lymph node metastases, but this trend was not statistically significant. Expression of CD44 splice variants containing epitopes encoded by exon v6 in primary tumors and pelvic lymph node metastases of cervical cancer patients is consistent with a prominent role of CD44 in the process of metastasis formation. © 1996 Wiley-Liss, Inc.     

CD44V6在乳腺癌中的表达及其临床意义

目的探讨乳腺癌中CD44v6蛋白的表达及临床意义.方法用免疫组织化学方法对乳腺癌CD44v6蛋白送行检测,并与患者临床病理指标进行分析.结果乳腺癌CD44v6蛋白阳性反应率为58.4%(28/48),与肿瘤大小、腋淋巴结转移成正相关.结论CD44v5参与了乳腺癌的发生、发展过程,可能是评估乳癌生物学行为的不良指标.     

CD44分子与乳腺癌

CD44分子属于黏附分子家族中的一员，其CD44的异常表达与乳腺癌的发生、发展和转移密切相关。近年来研究表明乳腺癌干细胞表面高度表达CD44分子，针对CD44分子与乳腺癌干细胞关系的研究，将为乳腺癌的临床治疗提供新的靶点。     

CD44分子与乳腺癌

CD44分子属于黏附分子家族中的一员,其CD44的异常表达与乳腺癌的发生、发展和转移密切相关。近年来研究表明乳腺癌干细胞表面高度表达CD44分子,针对CD44分子与乳腺癌干细胞关系的研究,将为乳腺癌的临床治疗提供新的靶点。     

Prognostic importance of the expression of CD44 splice variants in oral squamous cell carcinomas

Considering squamous cell carcinomas (SCCs) of the oral cavity and oropharynx the molecular mechanisms underlying the infiltration and destruction of adjacent tissue as well as the metastatic spread are largely unknown. In this context, the detection of defective expression of cellular adhesion molecules in the tumour cells, e.g. CD44, might be important and correlated with prognosis. Paraffin-embedded tumour-tissue from 99 patients with primary oral and oropharyngeal SCC, additionally including corresponding lymph-node metastases in nine cases, was analysed for expression of the CD44 splice variants v4, v5, v6, v7, and v9 by means of immunohistochemistry. A diminution of at least one of the examined CD44 isoforms compared to the normal oral epithelium was observed in 39.4% of the squamous cell carcinomas. No correlations could be found between CD44 expression and pT- or pN-stage. However, decreased expression of v9 was correlated with higher histological grade (p < 0.001). Moreover, reduced CD44 expression was a statistically significant independent predictor for shorter survival time (p = 0.002) as well as shorter recurrence-free interval (p = 0.004) in addition to pT- and pN-stage. The separate analysis showed that particularly the decreased v7 (p = 0.04) and v9 (p < 0.02) expression in the tumour cells was associated negatively with survival.     

CD44 splice variants confer metastatic behavior in rats: homologous sequences are expressed in human tumor cell lines.

One of several splice variants of CD44 expressed in metastasizing cell lines of rat tumors has been shown to confer metastatic potential to the non-metastatic variant of a rat pancreatic carcinoma line (U. Günthert et al., Cell, 65: 13-24, 1991). The variant-specific rat CD44 sequences were used to detect RNA expression in human cell lines: in carcinoma lines from lung, breast and colon; and in keratinocyte lines. By polymerase chain reaction amplification, complementary DNAs encoding human homologues were isolated and sequenced. The largest splice variant has been found in a large cell lung carcinoma line and in keratinocyte cell lines. It carries at least 5 additional domains (exons) encoding a total of 338 amino acids in the membrane-proximal extracellular region of the standard CD44. Various alternative splice products have been detected in other human tumor cell lines. The distribution of CD44 splice variants is consistent with the speculation that they fulfill functions in only a few restricted differentiation pathways and that in tumor cells these pathways have been reactivated.     

Soluble CD44 v5 and v6 in serum of patients with breast cancer. Correlation with expression of CD44 v5 and v6 variants in primary tumors and location of distant metastasis

Primary breast cancers were shown to overexpress CD44 v5 and v6 at the plasma membrane. However, the clinical significance of this overexpression remains unclear. Overexpression of CD44 v5 and v6 in primary breast cancers was found to correlate with metastasis and poor prognosis by some investigators, yet this correlation could not be confirmed by others using different antibodies. In this study the influence of metastatic disease, the site of metastasis, and the amount of CD44 v5 and v6 expression in the primary tumor on serum levels of the soluble forms of CD44 v5 and v6 (sCD44 v5 and v6) in breast cancer patients was investigated. Soluble CD44 v5 and v6 serum levels were measured by enzyme linked immuno sorbent assay in a group of breast cancer patients who developed metastases in various organs and in another group of patients with single organ metastasis. For control, sCD44 v5 and v6 levels were measured in breast cancer patients who remained free of metastasis and in healthy blood donors. Expression of plasma membrane bound CD44 v5 and v6 in the primary tumors of the patients with metastasis in various organs was correlated to sCD44 v5 and v6 levels in serum. Furthermore the size of sCD44 v6 was analyzed by immunoblot using a monoclonal antibody directed against CD44 v6. When metastases were detected, sCD44 v5 and v6 serum levels were increased as compared to levels measured one month after tumor surgery in patients free of metastases (p = 0.0025 and p = 0.0004). Six of 19 and 6 of 20 patients had sCD44 v5 and v6 serum levels above a cut-off level of 85 and 275 ng/mL, respectively. In these cases expression of CD44 v5 and v6 in the primary cancers was also elevated. Low sCD44 v5 and v6 serum levels were associated with weak expression of CD44 v5 and v6 in the respective primary cancers. As shown by statistical analysis of sCD44 v5 and v6 levels in 57 patients with single organ metastases, elevated sCD44 v6 levels but not sCD44 v5 levels were associated with metastases in liver or bone (p = 0.0025). Immunoblot analysis of soluble CD44 proteins in serum revealed two CD44 v6 specific signals of approximately 120 and 170 kDa. Increased sCD44 v5 and v6 serum levels in patients with breast cancer were influenced by the amount of CD44 v5 and v6 expression in the primary tumor by the site of metastasis. Elevated sCD44 v6 serum levels were preferentially found in patients with metastases in liver or bone.     

Cytosol concentrations of CD44 isoforms in breast cancer tissue

The role of the adhesion molecule CD44 in the natural history of breast cancer is controversial. We investigated the CD44 isoform CD44v5 and CD44v6 concentrations in the cytosol of 90 breast cancer specimens, 9 fibroadenomas and 22 normal breast tissue specimens by means of ELISA. CD44v5 and CD44v6 cytosol concentrations were statistically significantly higher in breast cancer compared with fibroadenoma and normal breast tissue (Mann-Whitney U-test, p = 0.009 and p < 0.001, respectively). When CD44 isoforms were correlated with lymph node involvement, histological grading, histological type, tumor stage and age at diagnosis, we found no statistically significant correlation with any of the investigated clinico-pathological parameters. In univariate and multivariate analyses, CD44v5 and CD44v6 were of no prognostic relevance regarding disease-free survival in breast cancer patients (log-rank test, p = 0.16 and p = 0.08, respectively). Our results indicate that CD44 isoforms are increased in samples from tumors relative to normal tissue. Our data show that CD44v5 and CD44v6 isoform expression, although up-regulated by malignant transformation, is not required to acquire a metastatic phenotype in breast cancer. Furthermore, our data support the assumption that cytosolic CD44 isoforms are of no prognostic relevance for disease-free survival of breast cancer patients. Int. J. Cancer (Pred. Oncol.) 79:541–545, 1998.© 1998 Wiley-Liss, Inc.     

CD44 attenuates metastatic invasion during breast cancer progression

Metastatic invasion is the primary cause of breast cancer mortality, and adhesion receptors, such as CD44, are believed to be critical in this process. Historically, primary breast tumor epithelium has been investigated in isolation from other tissue components, leading to the common interpretation that CD44 and its primary ligand, hyaluronan, promote invasion. Here, we provide in vivo evidence showing CD44 antagonism to breast cancer metastasis. In a mouse model of spontaneously metastasizing breast cancer (MMTV-PyV mT), we found that loss of CD44 promotes metastasis to the lung. Localization studies, in combination with a novel hyaluronan synthase-GFP transgenic mouse, show a restricted pattern of expression for CD44 and hyaluronan. Whereas CD44 is expressed in tumor epithelium, hyaluronan synthase expression is restricted to stromal-associated cells. This distinct CD44 and hyaluronan pattern of distribution suggests a role for epithelial-stromal interaction in CD44 function. To define the relevance of this spatial regulation, we developed an in vitro invasion assay to emulate invasion into the extracellular matrix. Invasion of CD44-positive tumor cells was inhibited in hyaluronan-containing matrices, whereas blocking CD44-hyaluronan association increased invasion. Collectively, these data show that during breast cancer progression, hyaluronan-CD44 dynamics occurring through epithelial-stromal interactions are protective against metastasis.     

Expression of CD44s in human colorectal cancer

CD44s is a cell adhesion molecule, which belongs to the family of hyaluronan binding proteins. Anti-body to CD44s is used to establish the association of its expression with the clinicopathological characteristics of colorectal cancer using immunohistochemical methods. The aim of this study is to investigate the expression of the standard form of CD44 (CD44s) in colorectal cancer tissues as compared to adjacent normal colonic tissues. Furthermore, the level of expression of CD44s in colorectal cancer tissues was correlated with the degree of histological differentiation, Duke s classification, sex, size and site of the tumor. Immunohistochemical analysis for CD44s was carried out in 49 paraffin-fixed sections of neoplastic colorectal tissues and non-neoplastic ones adjacent to the lesion, by the standard peroxidase-antiperoxidase method. Expression of these antigens were compared in normal and malignant epithelium and stromal cells. The results show that the level of CD44s in the epithelial and stromal cells was significantly higher in the colorectal cancer tissues than the normal ones. However, there was no association between the percentages of expressions of CD44s and the degree of histological differentiation, Duke s classification, sex or size of the tumor. There was however, a significantly higher expression of CD44s in the epithelium of rectal cancer than that of colonic cancer. This study indicates that the expression of CD44s is significantly higher in colorectal cancer tissues. However, further studies are required to understand its role in tumor progression and metastasis of this disease.     

Influence of cytokines on the expression of fas ligand and CD44 splice variants in colon carcinoma cells.

The expression of Fas ligand (FasL) by malignant cells might be a mechanism for tumor immune escape. We investigated FasL expression by LS 174T colon carcinoma cells. Furthermore, the effects of in vitro stimulation with rIL-2, rIFN-gamma and rTNF-alpha were investigated with regard to a possible regulation of the FasL expression by cytokines. FasL expression was detected by flow cytometry and RT-PCR. We observed a spontaneous expression of FasL by LS 174T cells. Incubation with high-dose rTNF-alpha induced an upregulation of FasL of 23%. rIL-2 and rIFN-gamma did not significantly affect FasL expression. To control whether our cytokine stimulation experiments were suitable to prove an upregulation of membrane proteins by tumor cells, we investigated the expression of ICAM-1, N-CAM, CD44s, CD44v6 and CD44v10. These adhesion molecules were spontaneously expressed by LS 174T cells. Only ICAM-1 and CD44v10 were significantly upregulated by rIFN-gamma and rTNF-alpha, respectively. These results could indicate that cytokines, released by tumor-infiltrating leukocytes, may induce the FasL-dependent apoptotic signal by which tumors downregulate an immunological host response. Copyright Copyright 1999 S. Karger AG, Basel