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Evaluation of the Prognostic Value of Serum Soluble CD 44 in Patients with Breast Cancer
Abstract
The outcome of breast carcinoma is usually determined by multiple factors. Aberrant expression of the cell adhesion molecule CD 44 has been claimed to be associated with poor prognosis in various human malignancies. This study was designed to investigate any correlation between the soluble adhesion molecule CD 44 and the clinicopathologic variables and to evaluate the possible prognostic significance of soluble CD 44. Venous blood samples were preoperatively collected from 100 patients with invasive breast carcinoma. The serum levels of different soluble CD 44 molecules (CD 44 standard form and CD 44 splice variant V6) were measured with an enzyme immunoassay method. The data of primary tumor status, age, estrogen receptor status, lymph node status, histologic grading, distant metastases status, TNM staging, S-phase fraction, and ploidy pattern were collected and evaluated simultaneously with the serum levels of soluble CD 44 st and CD 44 V6. Twenty healthy subjects were used as the control group. The serum levels of soluble CD 44 st showed no significant elevation in patient group. The mean value of soluble CD 44 V6 in patient group was 269.2 +/- 94.3 ng/ml and that of the control group was 179.5 +/- 50.7 ng/ml; the difference was significant (p < 0.01). In multivariate analysis, distant metastasis (p < 0.05) and TNM staging (p < 0.01) appeared as independent factors regarding the significant higher serum levels of soluble CD 44 V6. Based on our preliminary results, preoperative serum soluble CD 44 V6 is closely related to distant metastases and TNM staging. The possible role of soluble CD 44 V6 in the prognostic value of breast carcinoma deserves further elucidation and evaluation with long-term patient follow-up.
... We observed that high serum CD44 levels were correlated with postmenopausal status, ER negativity, PR negativity and adjuvant chemotherapy. However, there have been studies showed that upregulations of serum CD44 were associated with larger tumor size, lymph node or distant metastasis and higher stage in breast cancer patients [27,28], that is different from ours. This may be caused by that the patients in our study were diagnosed of breast cancer with stage I-III but other studies included stage IV cases. ...
Background: CD44 and ALDH1 have been recognized as the most widely used markers to identify breast cancer stem cells (BCSCs). However, limited to tissue sample and rare population, BCSCs have always been not easily detected. We aimed to measure CD44 and ALDH1A1 (major contributor to ALDH1 activity) levels in serum and explore the prognostic value in primary breast cancer patients.
Methods: This study included 140 primary breast cancer patients with stage I-III. Serum samples were collected before surgery and stored at -80 degrees. CD44 and ALDH1A1 were measured by chemiluminescent assay.
Results: High serum CD44 levels (≥ 417.4 ng/mL) were correlated with postmenopausal status (P = 0.006), estrogen receptor negativity (P = 0.025), progesterone receptor negativity (P = 0.002) and adjuvant chemotherapy (P = 0.003). The mean serum CD44 levels of luminal group (406.4 ± 68.3 ng/mL) were significantly lower than triple negative group (506.8 ± 175.5 ng/mL) (P < 0.001). There was no correlation between serum ALDH1A1 levels and molecular subtypes. Multivariate analysis revealed that high serum CD44 level (≥ 417.4 ng/mL), was an independent factor for PFS (P = 0.019) and OS (P = 0.008). However, serum ALDH1A1 has no impact on either PFS (P = 0.613) or OS (P = 0.441).
Conclusion: Serum CD44 was an independent prognostic indicator in primary breast cancer. However, serum ALDH1A1 has no impact on survivals and might not be an appropriate candidate to predict prognosis for breast cancer.
... CD44 can be shed from the cell surface by proteolytic cleavage. Increased plasma levels of soluble CD44 (solCD44) have been observed in different malignancies (Sheen-Chen et al., 1999;Yamane et al., 1999;Stickeler et al., 2000;Yasasever et al., 2000;Molica et al., 2001;Niitsu and Iijima, 2002). Furthermore, studies with melanoma cell lines have demonstrated that the cell proliferation-promoting effect of HA can be abolished in vitro and in vivo by solCD44 (Ahrens et al., 2001b). ...
CD44 proteins are cell surface receptors for hyaluronic acid (HA), a component of the extracellular matrix that has multiple effects on cell behavior. CD44 can be shed from the cell surface by proteolytic cleavage. The resulting soluble form can interfere with the interaction between HA and membrane-bound CD44. Soluble CD44 can abolish the cell proliferation-promoting effect of HA on melanoma cell lines, suggesting that a better understanding of the shedding process might identify ways of blocking tumor cell proliferation. ADAM10, ADAM17, and MMP14 have previously been implicated in the shedding of CD44 from various tumor cells. Using immunohistochemistry we demonstrate that ADAM10 and ADAM17 but not MMP14 are significantly expressed on melanoma cells in histological sections. In human melanoma cell lines expression of these proteases could be blocked by transfection with appropriate siRNAs. However, only blocking of ADAM10 expression led to decreased shedding of CD44. In parallel, cell proliferation was promoted. Confocal microscopy demonstrated that ADAM10 and CD44 colocalize on the cell surface. We conclude that ADAM10 is the predominant protease involved in the constitutive shedding of endogenous CD44 from melanoma cells, and that enhancement of ADAM10 activity could be an approach to decrease the proliferation of melanoma cells.
... Indeed, following the interest generated by this work, v6 expression has been examined in many tumors. Expression of serum-soluble CD44 v6 has been evaluated in patients with breast cancer and has been found to be closely related to distant metastases and TNM staging, suggesting that it may be of prognostic value (Sheen-Chen et al., 1999). Overexpression of CD44v6 has also been correlated with poor prognosis in patients with osteosarcoma and colon adenocarcinoma (Koretz et al., 1995;Kuryu et al., 1999), although studies of this isoform in other tumors have produced equivocal or negative results (Salmi et al., 1993). ...
Cell adhesion molecules (CAMs) are found on the surfaces of all cells, where they bind to extracellular matrix molecules or to receptors on other cells. As well as having a structural role, CAMs function as signaling receptors, transducing signals initiated by cellular interactions which regulate many diverse processes, including cell division, migration, and differentiation. Cell adhesion molecules are essential for maintaining stable tissue structure. However, cell adhesion must be dynamic to facilitate the mobility and turnover of cells. In dynamic situations, cells alter their cell-cell and cell-matrix interactions by virtue of altered expression and function of CAMs. The expression of CAMs is normally tightly regulated, thereby controlling cell proliferation, mobility, differentiation, and survival. Many of these processes are misregulated in malignant tumors, and it has been shown that many of the characteristics of tumor cells are attributable to the aberrant expression or function of CAMs. Integrins and E-cadherin are the most important CAMs expressed by stratified squamous epithelium. Altered expression of these molecules has been found in oral carcinoma, where loss of CAM expression is often seen in poorly differentiated lesions. However, up-regulation of certain integrins, such as alphavbeta6, has consistently been found in oral cancer, suggesting that it may play an active role in disease progression.
... All of the cancer were graded according to the criteria described by Bloom and Richard (11). Estrogen receptor status was determined by immunohistochemical staining method (12)(13)(14)(15)(16)(17). Thirty-five patients with benign breast tumor (20 patients with fibrocystic disease and 15 patients with fibroadenoma) were used as control group. ...
Hepatocyte growth factor (HGF) has been reported the cause of many biological events, including cell proliferation, movement, invasiveness, morphogenesis, and angiogenesis. Elevated hepatocyte growth factor content in tumor tissue was reported to predict a more aggressive biology in non-small cell lung cancer patients. However, there is still limited knowledge about the role of HGF in breast cancer. This study was designed with the aim to elucidate the possible relationship between the preoperative circulating soluble HGF and breast cancer.
One hundred twenty-four consecutive patients with invasive breast cancer undergoing surgery were prospectively included and evaluated. Venous blood samples were collected before the surgery. Sera were obtained by centrifugation and stored at -70 degrees C until assayed. The control group consisted of 35 patients with benign breast tumor (20 with fibrocystic disease and 15 with fibroadenoma). Serum concentrations of soluble HGF were measured by the quantitative sandwich enzyme immunoassay technique. The data on primary tumor staging, age, estrogen receptor status, lymph node status, distant metastases status, histologic grading, and tumor-node-metastasis (TNM) staging were reviewed and recorded.
The mean value of serum soluble HGF in patients with invasive breast cancer was 529.05 +/- 123.33 pg/mL and that of control group was 343.00+/- 31.03 pg/mL and the difference was significant (P < 0.001). Furthermore, there were significantly higher serum levels of soluble HGF in patients with negative estrogen receptor (P = 0.035), in patients with poorer differentiated tumor (P < 0.001), in patients with more advanced primary tumor staging (P < 0.001), in patients with more advanced lymph node status (P < 0.001), in patients with distant metastases (P < 0.001), and in patients with more advanced TNM staging (P < 0.001). In multivariate analysis by the multiple linear regression method, TNM staging (P < 0.001) seemed an independent factor regarding the significant higher serum levels of soluble HGF.
Patients with more advanced TNM staging were shown to have higher serum soluble HGF. Thus, preoperative serum soluble HGF levels might reflect the severity of invasive breast cancer and deserve further evaluation.
... Variant exon 6 promotes metastasis of rat pancreatic and breast carcinoma cells (9), and CD44v6 protein expression is reported to be an adverse prognostic factor in human non-Hodgkin's lymphoma (10) and colorectal carcinoma (11). In breast cancer patients, increased serumsoluble CD44v6 levels have been found to correlate significantly with tumor stage, lymph node status, and the presence of metastasis (32). This suggests that CD44v6 isoforms may play a role in the progression of invasive breast carcinoma. ...
CD44 is a multifunctional cell surface receptor with many known splice variants, some of which have been reported to play a role in tumor progression. The purpose of this study was to evaluate the prognostic significance of CD44 isoforms in early-stage, lymph node-negative invasive breast carcinoma.
Immunohistochemical staining for CD44 isoforms was done on archival paraffin tissue sections of invasive breast carcinoma from a cohort of lymph node-negative patients who received no adjuvant tamoxifen or chemotherapy and who had a mean clinical follow-up period of 15 years. Immunohistochemical staining was done with antibodies to CD44s, the standard isoform of CD44, and to isoforms containing variant exon 6 (CD44v6); levels of staining were correlated with clinical outcome data.
There was a trend towards increased disease-free survival for patients whose tumors had high anti-CD44s positivity (P = 0.05), and a significant association was observed between anti-CD44s positivity and disease-related survival (P = 0.04). Expression of CD44v6 isoforms did not correlate with clinical outcome.
CD44 expression, as assessed by immunohistochemical staining with anti-CD44s, may be a favorable prognostic factor in patients with node-negative invasive breast carcinoma.
... All the patients underwent modified radical mastectomy due to invasive breast cancer, defined as carcinoma with invasion to or beyond the basement membrane regardless of histological classification (ductal or lobular) (25). The data of primary tumor staging, age, estrogen receptor status (26)(27)(28)(29)(30)(31), lymph node status, histological grading and TNM staging were also collected. The hematoxylin-eosin stained slides of the paraffin-embedded tumor specimens were reviewed by our pathologists to confirm the accuracy of the histological diagnoses and lymph node status. ...
Constitutively activated signal transducer and activator of transcription (STAT) proteins are found in various types of tumors. However, there is still very limited information about the role of STATs in breast cancer. The power of the tissue microarray analysis (TMA) technique is the capability of performing a series of analyses of thousands of specimens in a parallel fashion with minimal damage to the original blocks. This study was designed to use TMA in determing the STAT1 status in breast cancer tissues.
Archival tissue specimens from 102 patients with primary invasive breast cancer were selected and STAT1 expression was analyzed using immunohistochemical staining with tissue microarray. The data of primary tumor staging, age, estrogen receptor status, lymph node status, histological grading and TNM staging were also collected.
There were 18 patients (17.6%) with 0 expression in STAT1, 29 patients (28.4%) with 1 expression in STAT1, 21 patients (20.6%) with 2 expression in STAT1 and 34 patients (33.4%) with 3 expression in STAT1. There was no significant relationship between STAT1 expression and age (p = 0.203), estrogen receptor status (p = 0.221), histological grading (p = 0.861), primary tumor staging (p = 0.918), lymph node status (p = 0.53), or TNM staging (p = 0.826). There was no survival difference noted among the four groups with different STAT1 expression (p = 0.859).
Immuno-histochemical staining with tissue microarray analysis was convenient and feasible for the analysis of STAT1 expression status in breast cancer. STAT1 expression did not show significant correlation with the overall survival rate.
... All the patients underwent modified radical mastectomy due to invasive breast cancer, defined as carcinoma with invasion to or beyond the basement membrane regardless of histological classification (ductal or lobular) (12). The data regarding primary tumor staging, age, estrogen receptor status (13)(14)(15)(16)(17)(18), lymph node status, histological grading and TNM staging were also collected. The hematoxylineosin-stained slides of the paraffined-embedded tumor specimens were reviewed by our pathologists to confirm the accuracy of the histological diagnoses and lymph node status. ...
Recent reports on the risk of prostate, breast, colorectal and lung cancer suggested that high circulating insulin-like growth factor-1 (IGF-1) concentrations are associated with an increased risk of cancer. The power of the tissue microarray (TMA) technique is the ability to perform a series of analyses of thousands of specimens in a parallel fashion with minimal damage to the original blocks.
Archival tissue specimens from 106 patients with primary invasive breast cancer were selected and IGF-1 expression was analyzed by immunohistochemical staining of tissue microarrays. The data regarding primary tumor staging, age, estrogen receptor status, lymph node status, histological grading and TNM staging were also collected.
There were 2 patients (2%) with grade 1 expression for IGF-1, 39 patients (37%) with grade 2 expression and 65 patients (61%) with grade 3 expression. There was no significant relationship between IGF-1 expression and age (p=0.256), estrogen receptor status (p=0.921), histological grading (p=0.815), primary tumor staging (p=0.455), or TNM staging (p=0.194). No survival difference was noted among the three groups with different IGF-1 expression (p=0.462).
Immunohistochemical staining of the tissue microarray was convenient and feasible for the analysis of IGF-1 expression in breast cancer, yet its expression did not show any significant correlation with the overall survival rate.
Once a diagnosis of cancer is established, the urgent question is whether it is localized or has already spread to regional lymph nodes and visceral organs. Despite improvements in early diagnosis, surgical techniques, general patient care and local and systemic adjuvant therapies, most deaths of cancer patients result from the relentless growth of metastatic disease that is resistant to conventional therapies. Surgical excision of primary neoplasms is not curative in many patients because by the time of diagnosis, metastasis may well have occurred. The major challenge for treatment of metastasis is the biological heterogeneity of cancer cells within primary lesions and especially within metastases. A wide range of genetic, biochemical, immunological and biological characteristics that differ from subpopulation to subpopulation include the display of cell-surface receptors, enzymes, karyotypes, cell morphologies, growth properties, sensitivities to various therapeutic agents and the ability to invade and produce metastasis [1,2]. This heterogeneity is due to differences in etiology, origin and selection pressures. The outcome of the metastatic process depends on the interaction of metastatic cells with host homeostatic factors, which include various elements of the host immune system. The establishment and progressive growth of metastatic cells requires that they exploit, subvert and suppress, when necessary, natural host defense systems. This chapter will focus on the host-tumor interactions that result in immune modulation of tumor cell populations.
The second part of the two-part review on breast cancer biomarkers, will consider information on new developments in diagnosis and treatment of breast cancer, which are inherently related to the result of scientific achievements in molecular and cell biology. This overview describes current knowledge, dominant trends and problems related to biomarkers in breast cancer. It stresses the importance of protein markers: cyclines, proteins of invasiveness i. e. aspartile, serine, cysteine and metalloproteinase, DNA repairing processes related to methylation; as well as adhesive proteins: E-cadherine/catenine complex, CD44, and integrines. Other biomarkers such as CHEK2, encoding phase G2 kinase - playing role in DNA repairing - and polymorphic epithelial mucines MUC1 and MUC2, as well as drug resistance markers are also described. The overview deals with problems related to the development of verifiable biological markers due to technical and methodological obstacles and, amongst other low concentrations of cancer proteins, negligible structural differences between cancerous and healthy subject proteins, and lack of tissue and organ marker specificity, required for primary cancer site identification.
Tumor cell migration is a multiple step process that involves concerted action of adhesion molecules and proteases outside the cells. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is believed a key enzyme in this process, and a cell surface hyaluronan receptor CD44 is reported to be one of the substrates of this enzyme. Here many migrative tumor cells are shown to express CD44 and MT1-MMP. Highly migrative fibrosarcoma cell line HT1080 endogenously expressed these two molecules. The migration of HT1080 cells was inhibited by a synthetic MMP inhibitor BB94, specific downregulation of MT1-MMP, and by expressing mutants of MT1-MMP or CD44, which interrupts the association between them. And the processing of CD44 was partially inhibited. These results address the possibility that the processing of CD44 by MT1-MMP plays a crucial role in CD44-mediated tumor cell migration in HT1080 cells.
The tumor secretion markers used most in the daily practice are reviewed, emphasizing the physiological aspects having great practical usefulness. Likewise, the possible value of other recently described substances which could be introduced into the daily practice in the next future is explained.
Hepatocellular tumors are pathologically divided into a limited number of entities such as focal nodular hyperplasia, hepatocellular
adenoma, hepatocellular carcinoma and its variants, and hepatoblastoma. Recent advances in immunophenotypic and molecular
characterization have led to an increased appreciation of the complexities of these growths. For example, subtypes of hepatocellular
adenomas with differing premalignant potentials have been defined, our ability to differentiate hepatocellular carcinoma from
high-grade dysplasia continues to improve, and molecular similarities of histologically discordant elements of combined hepatocellular/cholangiocellular
carcinoma have been reported. This chapter describes pathologic, immunophenotypic, and molecular features of hepatocellular
tumors. Continued progress in our understanding of these growths at the cellular and subcellular levels suggests that categorization
of these tumors may continue to evolve as additional significant clinicopathologic correlates are discovered.
Cell adhesion molecules (CAMs) arefound onthesurfaces ofall cells, wheretheybindtoextracellular matrix molecules ortoreceptors onother cells. Aswell ashaving astructural role, CAMsfunction assignaling receptors, transducing signals initiated bycellular interactions whichregulate manydiverse processes, including cell division, migration, anddiffer- entiation. Cell adhesion molecules areessential formaintaining stable tissue structure. However, cell adhesion mustbedynam- ictofacilitate themobility andturnover ofcells. Indynamic situations, cells alter their cell-cell andcell-matrix interactions by virtue ofaltered expression andfunction ofCAMs.Theexpression ofCAMsisnormally tightly regulated, thereby controlling cell proliferation, mobility, differentiation, andsurvival. Manyofthese processes aremisregulated inmalignant tumors, andit hasbeenshownthat manyofthecharacteristics oftumorcells areattributable totheaberrant expression orfunction ofCAMs. Integrins andE-cadherin arethemostimportant CAMsexpressed bystratified squamous epithelium. Altered expression of these molecules hasbeenfound inoral carcinoma, whereloss ofCAMexpression isoften seeninpoorly differentiated lesions. However, up-regulation ofcertain integrins, suchasav16, hasconsistently beenfound inoral cancer, suggesting that itmay playanactive role indisease progression.
Background:
Many studies have shown that focal adhesion kinase (FAK) is a positive regulator of tumor progression and invasion. However, there is still very limited information about the role of FAK in breast cancer. Tissue microarrays (TMA) can analyze thousands of tissue samples in a parallel fashion with minimal damage to the origin block. This study was designed with the application of TMA to analyze the FAK status in breast cancer.
Patients and methods:
Archival tissue specimens from 98 patients with primary invasive breast cancer were selected and FAK expression was analyzed by immunohistochemical staining with TMA. The data of primary tumor staging, age, estrogen receptor status, lymph node status, histological grading and TNM staging were also collected.
Results:
There were four patients (4.0%) with grade 1 expression in FAK, 41 patients (41.8%) with grade 2 expression in FAK and 53 patients (54.2%) with grade 3 expression in FAK. There was no significant relationship between FAK expression and age, estrogen receptor status, histological grading, primary tumor staging, lymph node status and TNM stage. By multivariate analysis, the TNM stage was found to be significantly related to the overall five-year survival rate (p<0.00001).
Conclusion:
Immunohistochemical staining with TMA is a convenient and feasible method. Unfortunately, our preliminary results fail to show meaningful prognostic value of FAK in breast cancer. A larger prospective study is warranted for further evaluation.
Type I receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR) and erbB2, have been implicated in mammary carcinoma growth and metastasis. Recent evidence suggests that type I receptor signaling may be mediated by the CD44 family of transmembrane glycoproteins that also have been implicated in mammary tumor progression. Here, the authors tested whether CD44, EGFR, and erbB2 interacted and colocalized with one another in four mammary carcinoma cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and MDA-MB-436) and in cytology samples obtained from patients with metastatic breast cancer. CD44 constitutively colocalized and coimmunoprecipitated with erbB2 and EGFR in all four mammary carcinoma cell lines. CD44 also colocalized with erbB2 and EGFR in all cytology samples expressing erbB2. CD44 colocalized with EGFR in cells from only 1 of 16 erbB2-negative cytology samples. These data indicate that CD44-EGFR-erbB2 protein complexes occur in a high proportion of metastatic mammary carcinomas and suggest that CD44-type I receptor colocalization may be a novel prognostic marker for aggressive mammary cancers.
CD44 is an adhesion molecule involved in tumor cell invasion and metastasis. The function of CD44 in breast cancer is not understood completely, or is its role as a predictive or prognostic factor. In this study, we tested for the hypothesis that the concentration of soluble CD44 (sCD44) in serum is correlated with clinicopathological factors, especially HER2, and survival in patients with breast cancer. We retrospectively identified 110 patients with breast cancer who had been treated at The University of Texas MD Anderson Cancer Center (MDACC) from September 2001 to May 2004. Sera were collected before definitive surgery in patients with stage I or II breast cancer, before initiation of neoadjuvant chemotherapy (if indicated) for patients with stage I-III breast cancer, and before initiation of systemic therapy in patients with stage IV breast cancer. sCD44 levels were determined using an enzyme-linked immunosorbent assay. The median age at diagnosis was 51 years (range, 28.6-87.1 years). sCD44 concentration was correlated with tumor stage (P = 0.0308). sCD44 serum concentration did not predict pathological response in patients treated with neoadjuvant chemotherapy. Among patients with distant metastases, sCD44 levels were significantly higher in patients with liver involvement than in patients with metastases at other sites. The overall survival rate did not differ between patients with high sCD44 concentration and patients with low sCD44 concentration. However, sCD44 concentration was a significant predictor of overall survival for patients with HER2-positive breast cancer, while no difference in overall survival rates was observed in patients with HER2-negative breast cancer. To the best of our knowledge, this is the first study to show an association between circulating sCD44 levels and survival in HER2-positive breast cancer patients. Our results suggest a role for sCD44 as a prognostic marker. Furthermore, sCD44 level may offer a new clinical therapeutic target in HER2-positive breast cancer.
Tissue microarray (TMA) allows the rapid immunohistochemical analysis of thousands of tissue samples in a parallel fashion. This study was designed to analyze the cortactin status in breast cancer using TMA and to investigate the relationship of cortactin status to breast cancer biology.
Archival tissue specimens from 99 patients with primary invasive breast cancer were selected. The cortactin expression was analyzed by TMA. Age, estrogen receptor status, histological grading and TNM staging data were also collected.
There were 23 patients (23.2%) with low (+) expression of cortactin, 60 patients (60.6%) with intermediate (++) expression and 16(16.2%) with strong (+++) expression. There was no significant relationship between cortactin expression and age, histological grading, primary tumor staging, lymph node status, estrogen receptor and TNM stage. By multivariate analysis, estrogen receptor status and TNM staging were found to be significantly related to the overall five-year survival rate.
Cortactin expression failed to demonstrate a prognostic value for patients with breast cancer.
The creation of a tissue microarray (TMA) allows for the rapid immunohistochemical analysis of thousands of tissue samples in a parallel fashion. This study applied TMA in order to analyse the topoisomerase II alpha status in breast cancer and to elucidate its relationship in breast cancer biology.
Archived tissue specimens from 94 patients with primary invasive breast cancer were selected and the topoisomerase II alpha expression was analysed by TMA. Data concerning age, oestrogen receptor status, histological grading and TNM staging were also collected.
There were 20 patients (21.3%) with 1+ expression in topoisomerase II alpha, 33 patients (35.1%) with 2+ expression and 41 patients (43.6%) with 3+ expression. By multivariate analysis, oestrogen receptor status, histological grading and TNM staging were found to be significantly related to the overall five-year survival rate.
Topoisomerase II alpha expression failed to demonstrate prognostic value in patients with breast cancer.
El gen de la mamoglobina codifica para una proteína de 10 Kda, que en tejidos humanos adultos tan solo ha sido detectada en la mama. Nuestro objetivo ha sido la detección del gen h-MAN en muestras tumorales y la correlación de dicha expresión con parámetros biológicos de primera generación (variedad histológica, grado histológico y nuclear, e invasción ganglionar), segunda generación (receptores de estrógenos y progesterona) y tercera generación (p53, Ki67 y c-erbB-2), así como con la ploidía y el contenido de DNA, y marcadores de angiogénesis (VEGF) y apoptosis (Bcl-2 y Bax)
To determine the levels of serum sCD44v6 in patients with oral cancer and evaluate the value of serum sCD44v6 in adjuvant diagnosis, staging and monitoring treatment response in these patients.
A total of 112 hospitalized patients with oral and maxillofacial malignancy and 28 healthy individuals were examined for serum sCD44v6 levels. Venous blood was collected from these patients and the healthy individuals. One week after treatment, venous blood was collected once again in 60 patients with oral and maxillofacial squamous cell carcinoma (OSCC).
The sCD44v6 concentration was not significantly different between patients with oral and maxillofacial malignancy and control group (P > 0.05). The levels of serum sCD44v6 in patients with OSCC and salivary carcinoma showed no difference with those in control group (P > 0.05). The sCD44v6 level in patients with stage III and IV disease was higher than that of patients with stage I and II and that of the control group, but the difference was not significant (P > 0.05). Serum sCD44v6 levels in patients with OSCC after treatment became lower than that prevailed during pretreatment (P < 0.05).
The possible roles of CD44v6 in the diagnosis of oral and maxillofacial malignancy deserve further elucidation and evaluation. Serum sCD44v6 may be a valuable marker in monitoring treatment response in patients with OSCC.
IGFBP-3 has been reported to be growth-stimulatory. The creation of tissue microarray (TMA) allows for the rapid immunohistochemical analysis of thousands of tissue samples in a parallel fashion. This study was designed with the application of TMA to analyze the IGFBP-3 status in breast cancer with the hope to elucidate the possible relationship between IGFBP-3 expression and breast cancer biology.
Archival tissue specimens from 97 patients with primary invasive breast cancer were selected. The IGFBP-3 expression was analyzed by TMA. The data of primary tumor staging, age, estrogen receptor status, lymph node status, histological grading and TNM staging were also collected.
There were 40 patients (41%) with 0-1 expression of IGFBP-3, 52 patients (54%) with 2 expression of IGFBP-3 and 5 patients (5%) with 3 expression in IGFBP-3. By multivariate analysis, the IGFBP-3 expression turned out to be significantly related to the overall five-year survival rate.
The preliminary results about IGFBP-3 expression in breast cancer are intriguing and require further evaluation.
Constitutively activated signal transducers and activators of transcription (STAT) proteins are found in various types of tumors. However, there is still very limited information about the role of STATs in breast cancer. The power of tissue microarray technique is the capability of doing a series of analyses of thousands specimens in a parallel fashion with minimal damage to the origin blocks. This study was designed with the application of tissue microarray to analyze the STAT3 status in breast cancer.
Archival tissue specimens from 102 patients with primary invasive breast cancer were selected, and STAT3 expression was analyzed by immunohistochemical staining with tissue microarray. The data of primary tumor staging, age, estrogen receptor status, lymph node status, histologic grading, and tumor-node-metastasis staging were also collected.
By multivariate analysis, the STAT3 expression turned out to be significantly related to the overall 5-year survival rate (P = 0.024).
Immunohistochemical staining with tissue microarray was convenient and feasible for the analysis of STAT3 expression status in breast cancer. Our preliminary results are promising and deserve further evaluation.
We provide here an example of clinical application of functional glycoproteomics for cancer diagnosis. Sialyl Lewis a and sialyl Lewis x glycotopes, which are the specific ligands for selectins, and variant forms of CD44, which are the adhesion molecules recognizing hyaluronate, are both implicated in cancer metastasis. The CD44 variants modified by the sialyl Lewis a and sialyl Lewis x glycotopes are expected to have dual functions, serving as ligands for vascular selectins, and simultaneously having binding activity to vascular bed hyaluronate, and are expected to figure heavily in cancer metastasis. We developed a heterogeneous sandwich assay system to detect soluble CD44v specifically modified by the cancer-associated sialyl Lewis a/x glycotopes, using the extracellular domain of CD44v cleaved by the metalloproteinase ADAM10 as standard molecules. We also developed the assay system for CD44v modified by normal epithelial glycotopes including disialyl Lewis a and sialyl 6-sulfo Lewis x. The results indicated that serum levels of soluble CD44v modified by cancer-associated glycotopes were frequently increased in patients with cancers, while those of CD44v modified by the nonmalignant glycotopes tended to be elevated in patients with benign disorders.
There is some evidence to suggest that smoking may affect circulating levels of CD44 (sCD44) molecules. Therefore, we investigated the effect of smoking on the circulating level of sCD44 by comparing the change in total sCD44, sCD44v5, and sCD44v6 concentrations over 1 year in a group of people who quit smoking (n = 30) and a control group of people who continued to smoke (n = 30). Smoking status and compliance were monitored by analysis of plasma cotinine and expired CO levels and also by self-reported tobacco use. We show a dose-dependent relationship between smoke intake and baseline plasma concentrations of reputed tumor-associated CD44 variant isoforms (sCD44v5 and sCD44v6) in smokers (n = 60). There was a significant decline in the level of both sCD44v5 and sCD44v6 in quitters as compared with continuing smokers [-13.2 (95% confidence interval, -7.6 to -18.8; P < 0.001) and -62.2 ng/ml (95% confidence interval, -33.9 to -90.6; P < 0.001), respectively], but not in the total sCD44 concentration. These results show that the increased concentrations of sCD44v5 and sCD44v6 in smokers are dose related and reversible and suggest that the attributed diagnostic specificity and prognostic value of sCD44 molecules in malignant and inflammatory disease may be affected by smoking status.
Transforming growth factor beta1 (TGF-beta1) may be related to breast cancer progression.
Prospective study.
University hospital.
Sixty consecutive patients with invasive breast cancer undergoing surgery were prospectively included and evaluated. The control group consisted of 14 patients with benign breast tumors (7 with fibrocystic disease and 7 with fibroadenoma).
Venous blood samples were collected before the surgery. Sera were obtained by centrifugation and stored at -70 degrees C until assayed. Serum concentrations of TGF-beta1 were measured by quantitative sandwich enzyme immunoassay. Data on primary tumor stage, age, estrogen receptor status, lymph node status, distant metastases, and TNM staging (according to the Union Internationale Contre le Cancer) were reviewed and recorded.
Measurements of preoperative serum TGF-beta1 levels in patients with breast cancer.
The mean +/- SD value of serum TGF-beta1 in patients with invasive breast cancer was 498.7 +/- 249.7 pg/mL and in the control group was 495.2 +/- 225.5 pg/mL (P =.96). However, there were significantly higher serum levels of TGF-beta1 in patients with more advanced lymph node status (P =.04), more advanced TNM stage (P =.005), and poorer histological grade (P =.02). In multivariate analysis, TNM staging (P =.02) was demonstrated to be the independent factor related to significantly higher serum levels of TGF-beta1.
Patients with more advanced TNM stages were shown to have higher serum TGF-beta1 levels. Thus, serum TGF-beta1 levels may reflect the severity of invasive breast cancer.
Matrix metalloproteinases (MMPs) have been reported to be associated with invasive and metastatic behaviors of human malignant tumors. However, there is still limited knowledge about the role of matrix metalloproteinases-2 (MMP-2) in breast cancer. This study was designed with the aim to elucidate the possible relationship between the preoperative circulating MMP-2 and breast cancer. Fifty-seven consecutive patients with invasive breast cancer undergoing surgery were prospectively included and evaluated. Venous blood samples were collected before the surgery. Sera were obtained by centrifugation, and stored at -70 degrees C until assayed. The control group consisted of 12 patients with benign breast tumor (six with fibrocystic disease and six with fibroadenoma). Serum concentrations of MMP-2 were measured by the quantitative sandwich enzyme immunoassay technique. The data on primary tumor stage, age, estrogen receptor, lymph node status, and TNM staging were reviewed and recorded. The mean value of serum MMP-2 in patients with invasive breast cancer was 694.3+/-140.5 ng/ml and those of control group were 593.3+/-134.0 ng/ml and the difference was significant (P=0.026). Furthermore, there were significantly higher serum levels of MMP-2 in the patients with more advanced primary tumor staging (P=0.005), in the patients with more advanced lymph node status(P=0.011) and in the patients with more advanced TNM staging (P<0.001). In multivariate analysis, TNM staging (P<0.001) appeared as independent factor regarding the significant higher serum levels of MMP-2. Patients with more advanced TNM staging were shown to have higher serum MMP-2 levels. Thus preoperative serum MMP-2 levels might reflect the severity of invasive breast cancer and deserve further evaluation.
Type I receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR) and erbB2, have been implicated in mammary carcinoma growth and metastasis. Recent evidence suggests that type I receptor signaling may be mediated by the CD44 family of transmembrane glycoproteins that also have been implicated in mammary tumor progression. Here, the authors tested whether CD44, EGFR, and erbB2 interacted and colocalized with one another in four mammary carcinoma cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and MDA-MB-436) and in cytology samples obtained from patients with metastatic breast cancer. CD44 constitutively colocalized and coimmunoprecipitated with erbB2 and EGFR in all four mammary carcinoma cell lines. CD44 also colocalized with erbB2 and EGFR in all cytology samples expressing erbB2. CD44 colocalized with EGFR in cells from only 1 of 16 erbB2-negative cytology samples. These data indicate that CD44-EGFR-erbB2 protein complexes occur in a high proportion of metastatic mammary carcinomas and suggest that CD44-type I receptor colocalization may be a novel prognostic marker for aggressive mammary cancers.
It has been suggested that circulating soluble Fas (sFas) contributes to tumor progression. However, little is known about the role of sFas in breast cancer. This study was designed with the aim of elucidating the possible relation between sFas and breast cancer. A series of 57 consecutive patients with invasive breast cancer undergoing surgery were prospectively included in the study and evaluated. Venous blood samples were collected before surgery. Sera were obtained by centrifugation and stored at -70 degrees C until assayed. The control group consisted of 12 patients with benign breast tumors (6 with fibrocystic disease, 6 with fibroadenoma). Serum concentrations of sFas were measured by the quantitative sandwich enzyme immunoassay technique. The data on primary tumor staging, age, estrogen receptor status, lymph node status, tumor grading, and TNM staging were reviewed and recorded. The mean value of circulating sFas in patients with invasive breast cancer was 794.2 +/- 183.0 pg/ml and that of the control group 582.1 +/- 62.8 pg/ml; the difference was significant (p < 0.001). Furthermore, there were significantly higher serum levels of sFas in the older patients (age > or = 50) (p = 0.020) and in those with a more advanced TNM stage (p = 0.021). In the multivariate analysis, TNM stage (p = 0.005) appeared to be an independent factor for significantly higher circulating sFas in patients with invasive breast cancer. Thus circulating sFas levels may reflect the severity of invasive breast cancer. Hence the possible prognostic value of sFas for breast cancer deserves further elucidation and evaluation with long-term patient follow-up.
In this second part of the two-part review of breast cancer biomarkers and molecular medicine, the first section will consider additional breast cancer prognostic factors, including oncogenes, tumor suppressor genes, cell adhesion molecules, invasion-associated proteins and proteases, hormone receptor proteins, drug resistance proteins, apoptosis regulators, transcription factors, telomerase, DNA repair and methylation and transcriptional profiling using high-density genomic microarrays. The second section will consider the prediction of therapy response using the techniques of pharmacogenetics and pharmacogenomics.
Standard CD44 (CD44st), CD44 variant 5 (CD44v5), and CD44 variant 6 (CD44v6), intercellular adhesion molecule 1(ICAM-1), and vascular cell adhesion molecule 1(VCAM-1) are expressed in human malignant cells and tissues. Their mechanism remains unclear but has been reported to be associated with the progression and metastasis of malignancies.
In this study, we investigated any correlations between the soluble adhesion molecule CD44 (st, v5, and v6), ICAM-1, and VCAM-1 and the clinicopathologic variables (eg, age, sex, histological grading, tumor size, lymph node status, distant metastasis, and TNM staging) and evaluated the difference between the pretreatment level in the patients with head and neck cancer and that in the control group. Furthermore, we examined the difference between the pretreatment serum levels and the after-treatment serum levels in the group with head and neck cancer. The pretreatment and after-treatment serum levels of soluble CD44st, CD44v5, CD44v6, ICAM-1, and VCAM-1 were measured in 81 patients with head and neck cancer and in 20 healthy volunteers as controls.
There were no significant differences between the serum levels of sCD44st, sCD44v5, sCD44v6, sICAM-1, and sVCAM-1 and the clinicopathologic variables in cancer patients. However, the higher serum level of sCD44v6 was significantly associated with distant metastasis (P = .02). Especially, we found that the pretreatment serum levels of sCD44st, sCD44v5, and sCD44v6 were markedly associated with TNM staging (CD44st P = .0017, CD44v5 P = .0005, CD44v6 P = .0046). Furthermore, the median serum levels of sCD44st, sCD44v5, sCD44v6, sICAM-1, and sVCAM-1 before treatment of head and neck cancer were significantly higher than those of the control group (CD44st P = .0067, CD44v5 P = .0048, CD44v6 P = .0007, ICAM-1 P = .0089, VCAM-1 P = .0178). The median serum level of sCD44st after treatment in the group of patients was significantly lower than that of pretreatment (CD44st P = .0001). And, both the median serum levels of sCD44v5 and sCD44v6 after treatment were also lower than those of pretreatment (CD44v5 P = .0004, CD44v6 P = .0025).
The possible roles of soluble adhesion molecules in the prognosis of head and neck carcinoma deserve further elucidation and evaluation with long-term patient follow-up.
ADAM-17 (a disintegrin and metalloproteinase 17) is a membrane-anchored protein, which can cleave the ectodomain in a variety of transmembrane proteins. In the in vitro experiments with tumor cells, ADAM-17 is reported to cleave CD44, an adhesion molecule that interacts with hyaluronic acid, to promote tumor cell migration. In the present study, we examined ADAM-17 expression and CD44 cleavage in specimens from 50 patients diagnosed to have oral squamous cell carcinoma (SCC). Each specimen was divided into two pieces, one was studied by immunohistochemistry and the other was subjected to a Western blot. By coordinating the results of both analyses, ADAM-17 expression was evaluated to be high in 23 cases (46%). When CD44 cleavage was also studied by immunohistochemical staining as well as with Western blotting, CD44 cleavage was judged to be positive in 29 cases (58%). When the ADAM-17 expression level was compared with the CD44 cleavage state, most of the cases expressing high levels of ADAM-17 (87%) showed positive CD44 cleavage. The level of ADAM-17 expression was significantly correlated to the nodal metastasis and local recurrence in oral SCC. Our findings suggest that ADAM-17 is involved in CD44 cleavage and contributes to tumor progression in oral SCC.
CD44 is a cell surface glycoprotein involved in cell-cell and cell-substrate interactions, which may be shed or released into circulation by proteolytic enzymatic mechanisms. Alternative splicing of CD44 and aberrant levels of soluble CD44 variants in the serum of cancer patients have been correlated to tumor progression and metastasis in different tumors including breast cancer. In this study we evaluated the clinical value of CD44 serum levels (sCD44) in patients with primary breast cancer.
Concentrations of soluble isoforms sCD44std, sCD44v5 and sCD44v6 were determined with a sensitive ELISA and normalized against the total protein concentration (TP). Pre-operative serum samples from 82 patients and 67 age-matched healthy blood donors were analyzed. The results were correlated to clinico-pathological parameters (tumor size, grading, lymph node metastasis, etc.).
In sera of breast cancer patients, we detected elevated concentrations of sCD44v6 (P = 0.0001) and total protein TP (P = 0.0001) in comparison to healthy controls, whereas overall sCD44 (sCD44std) and sCD44v5 did not differ. Patients with sCD44v6-concentrations above the 75%-percentile showed an increased T stage (2.9 cm vs. 1.8 cm) as well as a higher risk for lymph node metastasis (55% vs. 35%). In breast cancer patients with lymph node metastasis the median value of sCD44v6 was significantly higher (P = 0.025) in comparison to patients without lymph node metastasis and healthy controls.
Our data suggest an upregulated expression of alternatively spliced soluble CD44 isoforms in breast cancer patients. The specific alterations of certain CD44 isoform concentrations (especially sCD44v6) may reflect disturbances of the nuclear splicing machinery in tumor cells. The clinical significance of our findings are underlined by the positive correlation of elevated sCD44v6 concentrations and lymph node metastases (r (s) = 0.25).
The WHO Histological Classification of Breast Tumors, published in 1968, has been completely revised. This second edition provides a recommended nomenclature, definitions, and code numbers for both tumors and tumor-like lesions. It aims at promoting uniformity in recording and reporting diagnoses in order to facilitate international and other comparisons.
The CD44 molecule is known to display extensive size heterogeneity, which has been attributed both to alternative splicing and to differential glycosylation within the extracellular domain. Although the presence of several alternative exons has been partly inferred from cDNA sequencing, the precise intron-exon organization of the CD44 gene has not been described to date to our knowledge. In the present study we describe the structure of the human CD44 gene, which contains at least 19 exons spanning some 50 kilobases of DNA. We have identified 10 alternatively spliced exons within the extracellular domain, including 1 exon that has not been previously reported. In addition to the inclusion or exclusion of whole exons, more diversity is generated through the utilization of internal splice donor and acceptor sites within 2 of the individual exons. The variation previously reported for the cytoplasmic domain is shown to result from the alternative splicing of 2 exons. The genomic structure of CD44 reveals a remarkable degree of complexity, and we confirm the role of alternative splicing as the basis of the structural and functional diversity seen in the CD44 molecule.
A new panel of mAbs was prepared to a stromal cell line known to support lymphocytes in Whitlock-Witte type long-term bone marrow cultures. These antibodies were then screened with a cell adhesion assay and four were selected that inhibited the binding of B lineage cells to stromal cell monolayers. Immunofluorescent and biochemical analyses revealed that these new antibodies detected epitopes of the previously described Pgp-1/CD44 antigen complex. Addition of Pgp-1/CD44 antibodies to Dexter-type long-term bone marrow cultures completely prevented emergence of myeloid cells and they also blocked lymphocyte growth in Whitlock-Witte type cultures. mAbs MEL-14, LFA-1, and CD45R did not inhibit under the same conditions and there was no apparent relationship to Ig isotype. Adherent layers in treated cultures were not unusual in terms of morphology and the antibodies did not affect factor-dependent replication of lymphoid or myeloid progenitor cells. Therefore, the mechanism of inhibition may not involve direct toxicity to precursors or microenvironmental elements. Previous studies in humans and mice have implicated Pgp-1/CD44-related glycoproteins in the migration of peripheral lymphoid cells, as well as interactions of cells with the extracellular matrix. These findings suggest that they may also be critical for formation of lymphoid and myeloid cells within bone marrow.
It is proposed that tumor cell instability and the expression of cellular diversification mechanisms ensure that malignant neoplasms contain heterogeneous, phenotypically diverse tumor cell subpopulations. In such potentially unstable cellular mixtures of tumor cell phenotypes, some malignant cells may ultimately evolve with the most favorable properties for their progression to metastatic cells. Rates of cellular phenotypic instability and phenotypic diversification as well as their underlying causes appear to vary greatly among different tumor cells, and they are probably modulated by further genetic and chromosome changes and more frequently by intra- and extracellular epigenetic events that also differ, depending on the nature of the tumor cells and their cellular and microenvironmental interactions. Diversified malignant cells are characterized by quantitative and perhaps a few qualitative differences in gene expression, which may explain their abilities to undergo rapid changes in phenotypic properties. As tumor diversification and selection proceed uniquely in vivo, highly malignant cell subpopulations may eventually become dominant and gradually and independently lose their cellular and microenvironmental responsiveness. Tumor cell diversification mechanisms may be similar or identical to normal developmentally regulated diversification mechanisms that are used during embryonic and postembryonic cell diversification and development.
The distribution in living cells of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been studied by use of monoclonal antibodies. The presence of the molecule throughout the plasma membrane and on the substrate attached surface of the cell was demonstrated by immunofluorescence. Cell growth kinetics were not altered and the cells remained motile in the presence of the antibody. The uniform distribution of the direct immunofluorescence stain persisted for long periods (greater than 100 h), which indicates that the fluorescent monoclonal antibodies may be used to trace antigen surface distribution during cell functions. In motile cells, but not G0 or confluent cells, the degree of fluorescent staining decreased toward the leading edge; this gradient increased markedly during the time that the antibody was bound to the cells. However, the gradation was not seen with the lipid probe, dihexadecylindocarbocyanine. The antigen was "patched" only by the application of a second antibody directed to the rat monoclonal antibody and the relationships of these patches to the underlying cytoskeleton were characterized.
Soluble CD44 is present in the serum of normal individuals (2.7 +/- 1.1 nM). The concentration of soluble CD44 in the serum is elevated in patients with advanced gastric (24.2 +/- 9.8 nM) or colon cancer (30.8 +/- 11 nM). Serum CD44 concentration correlated with tumor metastasis and tumor burden. Surgical resection of tumors resulted in decreases in serum CD44 levels. By Western blot analysis, monoclonal anti-CD44 antibody reacted with a major protein with molecular weight between 130,000 and 190,000. In addition, two proteins with molecular weights of 72,000 and 80,000 can also be identified. Therefore, different CD44 isoforms may be present in the serum of cancer patients. Serum CD44 concentrations may be an indicator of tumor burden and metastasis in patients with malignant diseases.
CD44 is a transmembrane glycoprotein occurring in several isoforms with different extracellular regions. The various transcripts are encoded by one gene locus containing 20 exons, of which at least 10 can be alternatively spliced in nascent RNA. Isoforms encoded by the variant exons (termed CD44v) are highly restricted in their distribution in nonmalignant tissue as opposed to the standard form of CD44 (CD44s) abundant in many tissues. Specific variant isoforms containing exon 6v have been shown to render nonmetastatic rat tumor cells metastatic. Based on the prominent role in rat metastasis formation, CD44v isoforms were suggested to be involved in human tumor progression. Correlations between prognosis and expression of CD44v have been reported for gastric and colon carcinoma, for non-Hodgkin's lymphoma, and recently for breast carcinoma. We evaluated the expression of CD44 isoforms in node-positive (n = 119) and node-negative (n = 108) cases of breast carcinoma by immunohistochemistry using CD44v exon-specific mAbs. In a subset of 43 cases of high-risk patients, reverse transcription-PCR was used to determine the exon composition of the transcripts. Protein and RNA expression data were probed statistically for their correlation to survival of the patients and clinical risk factors. In contrast to recently published data (M. Kaufmann et al., Lancet, 345: 615-619, 1995), in our cohort disease-free and overall survival data did not indicate significant correlations with the expression of the analyzed isoforms in univariate and multivariate analyses. Comparison of CD44 protein expression with established clinical risk factors for survival such as tumor size (pT1+pT2) and histological grading revealed correlations with the presence of CD44s (P = 0.02 and P = 0.03, respectively) and CD44-9v (P = 0.05 for histological grading). Carcinoma tissues with elevated estrogen and progesterone receptor levels showed positive correlation with CD44-6v (P = 0.001), while a trend for significant coexpression of CD44s and CD44-9v isoforms was observed in estrogen receptor-positive tissues (P = 0.08 and 0.06, respectively). In breast cancer, CD44s, CD44-9v, and CD44-6v are apparently markers for cellular differentiation but not for tumor progression. Our data suggest that steroid hormone receptors may be associated with the in vivo expression of CD44-6v-containing isoforms in human mammary carcinoma.
CD44, a cell surface glycoprotein, is involved in lymphocyte trafficking from the blood to lymphatic tissues, and is of importance in dissemination of lymphoma. A variant form of CD44 that has additional amino acids in the common protein backbone (CD44v6) also seems to play a role in the metastatic dissemination of malignancies. We measured serum CD44 and CD44v6 in 34 patients with lymphoma and in healthy controls by dot blot assay. Small amounts of both CD44 (range, 10 to 80 ng/mL) and CD44v6 could be detected in sera of all controls. Serum CD44 was elevated in all patients with lymphoma before treatment (range, 70 to > 2,000 ng/mL, P < .0001), and CD44v6 was also slightly elevated. Serum CD44 levels correlated with response to treatment. Patients with complete response achieved similar CD44 levels as the controls, whereas those with progressive disease had increased serum CD44 levels. We conclude that both the standard and the variant form of CD44 are detectable in sera of healthy individuals, and that serum CD44 may be useful in monitoring treatment response in patients with lymphoma.
The WHO Histological Classifiction of Breast Tumors, published in 1968 has been completely revised. This second edition provides a recommended nomenclature, definitions and code numbers for both tumors and tumor-like lesions. It aims at promoting uniformity in recording and reporting diagnoses in order to facilitate international and other comparisons.
A new panel of mAbs was prepared to a stromal cell line known to support lymphocytes in Whitlock-Witte type long-term bone marrow cultures. These antibodies were then screened with a cell adhesion assay and four were selected that inhibited the binding of B lineage cells to stromal cell monolayers. Immunofluorescent and biochemical analyses revealed that these new antibodies detected epitopes of the previously described Pgp-1/CD44 antigen complex. Addition of Pgp-1/CD44 antibodies to Dexter-type long-term bone marrow cultures completely prevented emergence of myeloid cells and they also blocked lymphocyte growth in Whitlock-Witte type cultures. mAbs MEL-14, LFA-1, and CD45R did not inhibit under the same conditions and there was no apparent relationship to Ig isotype. Adherent layers in treated cultures were not unusual in terms of morphology and the antibodies did not affect factor-dependent replication of lymphoid or myeloid progenitor cells. Therefore, the mechanism of inhibition may not involve direct toxicity to precursors or microenvironmental elements. Previous studies in humans and mice have implicated Pgp-1/CD44-related glycoproteins in the migration of peripheral lymphoid cells, as well as interactions of cells with the extracellular matrix. These findings suggest that they may also be critical for formation of lymphoid and myeloid cells within bone marrow.
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We investigated the expression of CD44 isoforms containing variant exons v5, v6 and v7–8 in 115 human breast cancer specimens by means of immunohistochemistry. CD44 isoforms CD44v5, CD44v6 and CD44v7–8 were detected in 56% (n = 64), 24% (n = 28) and 15% (n = 17), respectively. In 36 specimens of axillary lymph node metastasis, expression of CD44v5, CD44v6 and CD44v7–8 was found in 94% (n = 34), 92% (n = 33) and 89% (n = 32), respectively. Five year survival rates with or without CD44v5 and CD44v6 expression were 71% versus 86% (log-rank test, P = 0.02) and 62% versus 81% (log-rank test, P = 0.001), respectively. For disease-free survival, expression of CD44v5, CD44v6 and CD44v7–8 showed a prognostic impact (log-rank test, P = 0.004, P = 0.0001 and P = 0.0001, respectively). However, multivariate analysis revealed that all investigated CD44 isoforms failed to be independent predictors of the patient's outcome.
A variant of the glycoprotein CD44 (CD44v) that shares sequences with variants causally involved in metastasis formation is transiently expressed on B and T lymphocytes and macrophages after antigenic stimulation and in the postnatal period. Antibodies to the variant hinder in vivo activation of both B and T cells. The observation that a protein domain that is expressed on CD44 and required for the lymphatic spread of tumor cells can catalyze an essential step in the process of lymphocyte activation supports the idea that metastasizing tumor cells mimic lymphocyte behavior.
Expression of the CD44 molecule was examined in a variety of human brain tumours, brain metastases and normal brain. Immunohistological staining with several CD44 antibodies demonstrated differential expression of the CD44 molecule among different brain tumour types. CD44 was strongly expressed in high-grade gliomas and weakly expressed in meningiomas, medulloblastomas and normal brain. Northern blot analysis revealed the presence of 3 major CD44 mRNAs of 1.6, 2.2, and 5.0 kb in glioblastomas and a mRNA of 5.6 kb in meningiomas. CD44 expression was also detected by flow cytometric analysis on cultured cells derived from a variety of human brain tumours including glioblastomas and meningiomas.
The monocyte-derived cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), are central regulators
of the immune response, but the physiologic stimuli for their release remain largely undefined. Engagement of three monocyte
glycoproteins, LFA-3, CD44, and CD45, by specific monoclonal antibodies immobilized on plastic induced TNF-alpha and IL-1
beta release. In addition, TNF-alpha was released when monocyte LFA-3 bound immobilized, purified CD2, which is its physiologic
receptor. Thus, a receptor-ligand interaction that mediates cell-cell adhesion can transmit the necessary signals for the
release of monokines.
Using a monoclonal antibody (MAb1.1ASML) raised against a surface glycoprotein of the metastasizing rat pancreatic carcinoma cell line BSp73ASML, cDNA clones have been isolated that encode glycoproteins with partial homology to CD44, a presumed adhesion molecule. In one of the clones, pMeta-1, the epitope marks an additional extracellular domain of 162 amino acids inserted into the rat CD44 protein between amino acid positions 223 and 247 (by analogy to human and murine CD44). The new variants are expressed only in the metastasizing cell lines of two rat tumors, the pancreatic carcinoma BSp73 and the mammary adenocarcinoma 13762NF; they are not expressed in the non-metastasizing tumor cell lines nor in most normal rat tissues. Overexpression of pMeta-1 in the nonmetastasizing BSp73AS cells suffices to establish full metastatic behavior.
The CD44 inhibitor Lutheran [In(Lu)]-related p80 molecule has recently been shown to be identical to the Hermes-1 lymphocyte homing receptor and to the human Pgp-1 molecule. We have determined the effect of addition of CD44 antibodies to in vitro activation assays of PBMC. CD44 antibodies did not induce PBMC proliferation alone, but markedly enhanced PBMC proliferation induced by a mitogenic CD2 antibody pair or by CD3 antibody. CD44 antibody addition had no effect upon PBMC activation induced by PHA or tetanus toxoid. CD44 antibody enhancement of CD2 antibody-induced T cell activation was specific for mature T cells as thymocytes could not be activated in the presence of combinations of CD2 and CD44 antibodies. CD44 antibody enhancement of CD2-mediated T cell triggering occurred if CD44 antibody was placed either on monocytes or on T cells. In experiments with purified monocyte and T cell suspensions, CD44 antibodies A3D8 and A1G3 augmented CD2-mediated T cell activation by three mechanisms. First, CD44 antibody binding to monocytes induced monocyte IL-1 release, second, CD44 antibodies enhanced the adhesion of T cells and monocytes in CD2 antibody-stimulated cultures, and third, CD44 antibodies augmented T cell IL-2 production in response to CD2 antibodies. Thus, ligand binding to CD44 molecules on T cells and monocytes may regulate numerous events on both cell types that are important for T cell activation. Given that recent data suggest that the CD44 molecule may bind to specific ligands on endothelial cells (vascular addressin) and within the extracellular matrix (collagen, fibronectin), these data raise the possibility that binding of T cells to endothelial cells or extracellular matrix proteins may induce or up-regulate T cell activation in inflammatory sites.
The study of cell surface molecules that are involved in interactions between immune and non-hematopoietic cells in various microenvironments is currently an area of great interest. One molecule that appears to be involved in multiple steps of normal immune cell function is now called CD44 and has been known previously as Pgp-1, In(Lu)-related p80, Hermes, ECM-III and HUTCH-I. Within the past year, the co-identity of all of these independently discovered molecules has become apparent, and the role of the CD44 molecule in T-cell activation has been discovered. In this review, Barton Haynes and his colleagues bring together numerous divergent lines of investigation on the CD44 molecule, review the many functional roles attributed to it, and present a unifying view of how, with numerous ligands, it may participate in several areas of normal immune cell function.
In this review, we focus upon the mechanistic, functional, and evolutionary aspects of alternative splicing. The discussion of mechanisms attempts to be exhaustive, drawing not only upon direct experimental investigations of alternatively spliced genes, but also upon relevant themes derived from the study of constitutive splicing
The distribution of lymphocytes among the tissues of the body is not random. Both the number and the representation of particular functional subsets of lymphocytes are carefully controlled, differing in each lymphoid organ or tissue in a manner that presumably reflects local immune requirements. The tissue distribution of lymphocytes is a function of lymphocyte class, of their previous history or stage of differentiation, and of their antigenic specificity. For example, those lymphocyte populations primarily responsible for humoral immunity (B cells) predominate in the spleen and in the gut-associated Peyer’s patches, whereas T cells, which are primarily regulatory and cytotoxic cells, are the major lymphocyte type in the peripheral lymph nodes and skin (Stevens et al. 1982; Streilein 1978). Functionally and antigenically defined T-cell subsets are also unequally distributed between mucosal and nonmucosal lymphoid tissues (Elson et al. 1979; Kraal et al. 1983). The distribution of certain effector and effector-precursor populations can be even more restricted: especially dramatic is the segregation of IgA- vs. IgG-expressing B cells. Surface IgA-bearing lymphocytes are highly represented in the mucosa-associated lymphoid organs, and the mucosal surfaces attract predominantly IgA-secreting plasma cells (Guy-Grand et al. 1974; reviewed by Lamm 1976). In nonmucosal sites, such as peripheral lymph nodes or the skin, IgA-bearing cells are rare, and most plasma cells secrete IgM or IgG. Lymphocytes can also segregate in vivo on the basis of antigen specificity: antigen-specific B and T cells are disproportionately represented in lymph nodes or spleen challenged with antigen (Kraal et al. 1982; Sprent 1980) and antigen-specific plasma cells accumulate in tissue sites of specific antigen deposition (Husband and Gowans 1978; Husband 1982).
A method has been developed that allows flow cytometry to be used for measuring the cellular DNA content of paraffin-embedded human tumors. Thick (i.e., 30 micron) sections were cut from tissue blocks using a microtome and dewaxed in xylene. The sections were then rehydrated by sequentially immersing them in 100, 95, 70, and 50% ethanol before finally washing in distilled water. Single cell suspensions were then prepared by incubation in 0.5% pepsin, pH 1.5, at 37 degrees C for 30 min. The cells were counted, washed, and stained with 1 microgram/ml 4',6'-diamidino-2-phenylindole for 30 min, and DNA content was measured using an ICP 22 flow cytometer. There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin. This method allows the retrospective study of archival material where the clinical outcome is already known, and it should, therefore, be particularly useful for determining the prognostic significance of abnormal DNA content measured by flow cytometry.
The expression of specific cell adhesion molecule CD44 isoforms (splice variants) has been shown to be associated with poor prognosis in human malignancies, such as breast cancer. We used three different variant exon sequence-specific murine monoclonal antibodies to epitopes encoded by exons v5, v6 or v7-v8 of human variant CD44, to study the expression of CD44 splice variants by immunohistochemistry in human stage III cervical cancer. We investigated 40 pretreatment punch biopsies of cervical cancer FIGO stage III. CD44 splice variants CD44v5, CD44v6 and CD44v7-8 were detected by means of immunohistochemistry in 90%, 55% and 25%, respectively. CD44 epitopes encoded by exon v5 were not correlated with prognosis. Expression of CD44 splice variants containing epitopes encoded by exon v6 were correlated with significantly poorer prognosis (Mantel test, P = 0.008). Five-year survival rates with or without CD44v6 expression were 20% versus 71%, respectively. Expression of CD44v7-8 was also correlated with significantly poorer overall survival (Mantel test, P = 0.02). Expression of CD44 splice variants containing epitopes encoded by exons v7-v8 and especially exon v6 is associated with significantly poorer prognosis in stage III cervical cancer patients.
CD44, a cell surface glycoprotein, is involved in lymphocyte trafficking from the blood to lymphatic tissues, and is of importance in dissemination of lymphoma. A variant form of CD44 that has additional amino acids in the common protein backbone (CD44v6) also seems to play a role in the metastatic dissemination of malignancies. We measured serum CD44 and CD44v6 in 34 patients with lymphoma and in healthy controls by dot blot assay. Small amounts of both CD44 (range, 10 to 80 ng/mL) and CD44v6 could be detected in sera of all controls. Serum CD44 was elevated in all patients with lymphoma before treatment (range, 70 to > 2,000 ng/mL, P < .0001), and CD44v6 was also slightly elevated. Serum CD44 levels correlated with response to treatment. Patients with complete response achieved similar CD44 levels as the controls, whereas those with progressive disease had increased serum CD44 levels. We conclude that both the standard and the variant form of CD44 are detectable in sera of healthy individuals, and that serum CD44 may be useful in monitoring treatment response in patients with lymphoma.
CD44 variants containing v6 confer metastatic potential to rat carcinoma cell-lines. In man, CD44v6 is increasingly expressed during colorectal tumour progression. In 68 colorectal carcinoma patients, survival analysis showed that CD44v6 expression in the tumours was associated with tumour-related death. In patients who had an apparently radical resection of their primary tumour, CD44v6 expression had prognostic value independent of Dukes' stage. CD44v6 expression may reflect propensity for metastasis after apparently curative surgery, making adjuvant therapy an option in these patients.
CD44 designates a group of closely related cell-surface proteins generated by alternative splicing. We have previously shown that splice variants carrying sequences encoded by exon v6 are preferentially expressed in metastatic animal cancer cell lines and that they confer metastatic behaviour on non-metastatic animal tumour cell lines. In this study we set out to assess the expression of CD44 epitopes specific for variant exon sequences in human breast cancer and their potential for determining prognosis. We used affinity-purified polyclonal sera and four monoclonal antibodies raised against the human homologues of CD44 variant exon sequences to investigate the presence of CD44 on 100 primary invasive breast tumours, 12 local recurrences, 18 lymph node metastases, and normal tissue controls. Whereas normal mammary ductal epithelial cells and cells derived from hyperplastic lesions do not express CD44 variant exons, expression of v3, v5, and v6 epitopes was found in most tumour samples. The DIII (exon v6) epitope was present in 84% of the primary tumours and in 100% of axillary lymph node metastases and local recurrences. The presence of these CD44 epitopes is correlated with poor overall survival. 15 patients with exon-v6-negative tumours had good survival compared with 76 patients with exon-v6-positive tumours (p = 0.005; log rank test). Multivariate analysis showed that the CD44 epitope encoded by exon v6 was a good marker for prognosis independent of progesterone receptor, lymph node status, tumour size, and grade.
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.
The serum-soluble interleukin-2 receptor has been claimed to be a marker of host biological response in patients with solid malignancies. This study was designed to evaluate the biological significance of the preoperative serum-soluble interleukin-2 receptor concentration in patients with invasive breast cancer.
Venous blood samples were collected from 66 patients with invasive breast carcinoma and the serum concentrations of soluble interleukin-2 receptor were measured with an enzyme immunoassay method. Data regarding maximum tumor diameter, age, estrogen receptor status, lymph node status, distant metastasis status, histologic grade, ploidy pattern and S-phase fraction were also collected and evaluated simultaneously with the serum concentration levels of soluble interleukin-2 receptor. Fifteen age-matched healthy subjects were used as a control group.
The mean value of serum-soluble interleukin-2 receptor in patients with invasive breast cancer was 621 +/- 145 units/ml and that of the control group was 308 +/- 59 units/ml; and the difference was significant (p = 0.000). With multivariate analysis, lymph node status (p = 0.000), distant metastasis status (p = 0.000) and maximum tumor diameter (p = 0.000) appeared as independent factors in regards to the significantly, higher serum concentrations of soluble interleukin-2 receptor.
Based on our preliminary results, the preoperative serum-soluble interleukin-2 receptor concentration is closely related to lymph node status, distant metastasis status and tumor diameter in invasive breast carcinoma. This may be an additional valuable predictive factor for the diagnosis of invasive breast cancer.
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