Since the discovery of circulating tumor cells in 1869, technological advances in the study of biomarkers from liquid biopsy have made it possible to diagnose disease in a less invasive way. Although blood-based liquid biopsy has been used extensively for the detection of solid tumors and immune diseases, the potential of urine-based liquid biopsy has not been fully explored. Advancements in technologies for the harvesting and analysis of biomarkers are providing new opportunities for the characterization of other disease types. Liquid biopsy markers such as exfoliated bladder cancer cells, cell-free DNA (cfDNA), and exosomes have the potential to change the nature of disease management and care, as they allow a cost-effective and convenient mode of patient monitoring throughout treatment. In this review, we addressed the advancement of research in the field of disease detection for the key liquid biopsy markers such as cancer cells, cfDNA, and exosomes, with an emphasis on urine-based liquid biopsy. First, we highlighted key technologies that were widely available and used extensively for clinical urine sample analysis. Next, we presented recent technological developments in cell and genetic research, with implications for the detection of other types of diseases, besides cancer. We then concluded with some discussions on these areas, emphasizing the role of microfluidics and artificial intelligence in advancing point-of-care applications. We believe that the benefits of urine biopsy provide diagnostic development potential, which will pave opportunities for new ways to guide treatment selections and facilitate precision disease therapies.

This study examined the association of CD44V6 expression in ovarian cancer. We recruited 38 patients with ovarian cancer, 23 with benign ovarian tumor, and 20 with normal ovaries using RT-PCR and western block analysis. Compared with normal ovaries, the expression of CD44V6 mRNA was significantly elevated in benign ovarian tumor and ovarian cancer. At the protein level, we found no significant differences in CD44V6 expression between normal ovarian tissue and benign ovarian tumor. However, the expression of CD44V6 in ovarian cancer was significantly elevated compared to normal ovaries and benign ovarian tumor. These results were supported by ELISA and western blot analysis. Immunohistochemistry showed that CD44V6 protein in ovarian cancer cells accumulated at high levels on the membrane of ovarian cancer cells. CD44V6 expression is closely associated with the tumorous transformation of ovarian tissue, suggesting that CD44V6 can promote the occurrence and progression of ovarian cancer.

Simple, rapid, and sensitive detection of CD44 is of paramount importance since it plays pivotal roles in tumor initiation, growth and metastasis. Herein, we describe a novel method for sensitive, visual and facile fluorescence detection of CD44 and CD44-mediated cancer cell imaging, using a probe based on cationic conjugated polymer (CCP)-PFEP and fluoresceinamine-hyaluronan (FA-HA). HA is an anionic natural glycosaminoglycan that can specifically bind to the overexpressed CD44 on various kinds of cancer cells. PFEP and FA-HA formed a complex through electronic interactions, resulting in a highly efficient fluorescence resonance energy transfer (FRET) from PFEP to FA-HA; moreover, the efficiencies of FRET correlated with the concentrations of CD44 because the specific binding of HA-CD44 would separate FA-HA away from PFEP. This method did not require laborious and expensive dual-labeling or protein-labeling needed in previously reported detection methods of CD44. Just mix the sample and test solution containing the PFEP/FA-HA complex, and the results allowed naked-eye detection by observing fluorescent color of solutions with the assistance of a UV lamp. Most importantly, the use of a conjugated polymer with excellent amplification property as well as the specific binding of HA-CD44 endowed this method with high sensitivity and specificity, making it applicable for reliable quantitative detection of CD44. Furthermore, the PFEP/FA-HA complex formed nanoparticles in aqueous solution, and the nanoparticles can be selectively taken up by MCF-7 cells (cancer cell) through the HA-CD44 interaction, thereby giving rise to a dual-color tumor-targeted imaging probe with good photostability. The development of this fluorescent probe showed promising potential to make a reliable and routine method available for early diagnosis of cancer.

Several screening methods for reducing the mortality rate of colorectal cancer (CRC) have been reported in recent decades. Fecal occult blood tests (FOBTs) are widely used for CRC screening and immunochemical FOBTs perform better than guaiac FOBTs; however, the sensitivity and specificity of immunochemical FOBTs remain unsatisfactory. To resolve this problem, novel fecal molecular methods based on fecal protein, DNA and RNA analyses have been developed. Regarding fecal proteins, several marker proteins indicating intestinal bleeding and cancer cell-specific proteins have been investigated. Regarding fecal DNA, numerous gene mutation and gene methylation analyses have been reported. Consequently, fecal DNA analysis was recommended as a CRC screening method in 2008. In addition, gene expression analyses of CRC-specific genes and miRNAs in fecal RNA have been investigated over the last decade. This review article summarizes molecular methods using fecal samples for CRC screening, focusing on reports within the last 5 years.

CD44 or hyaluronan receptor is a transmembrane receptor associated with aggressive tumour growth, proliferation, and metastasis. In normal physiology, this receptor has a crucial role in cell adhesion, inflammation, and repair processes. However, many tumour cells over-express this receptor and abuse it to become progressive and perpetual units. The article comments from common functioning of the CD44 receptor, to its diabolic multi-dimensional effects in promotion of malignant cells. It also illuminates the relations of CD44 endorsed processes with other biomolecular events in cancer progression. In an end, the review focuses comprehensively at ongoing researches to exploit the CD44 over-expression as a probable target in treatment, management, and diagnosis of malignancy.

We investigated changes in the gene expression profile in colon cancer in order to identify gene markers that may be useful in the management of this disease.

The Cancer Genome Anatomy Project was used to detect differences in gene expression between normal and cancer tissue. The overexpression of dipeptidase-1 (DPEP1) in cancer tissue was confirmed in a sample of 76 patients by real-time PCR. To identify the function of DPEP1, RNA interference (RNAi) was used to inactivate this gene in the colon cancer cell line. Immunohistochemical analysis was performed to characterize the pattern of DPEP1 expression in colon cancer.

DPEP1 expression in cancer was significantly higher than that in normal tissue. However, DPEP1 expression decreased with pathological differentiation, lymph-node and distant metastasis. Patients with tumors with decreased DPEP1 expression showed a poorer prognosis, and this was also true of patients with tumors who are treated with curative intent. RNAi-mediated DPEP1 reduction in the colon cancer cell line did not result in cell proliferation or apoptosis, but was associated with an increased invasive ability. DPEP1 protein was observed on the apical side of the cancer cells, and is expressed in the early stages of carcinogenesis, even in adenomas of both sporadic colorectal cancer and familial adenomatous polyposis patients.

DPEP1 expression in normal colonic mucosa is very low, but it is highly expressed in colorectal adenoma and cancer specimens and is negatively correlated with parameters of pathological aggressiveness and poor prognosis. DPEP1 is expressed in the early stages of colon carcinogenesis and affects cancer cell invasiveness.

Aim-To demonstrate the feasibility of studying specific gene expression in fine needle aspirates from clinical lesions. The reverse transcription/polymerase chain reaction (RT-PCR) technique was used to demonstrate CD44 gene expression in cells from diagnostic fine needle aspirates taken from patients attending the outpatient clinic for breast diseases.Methods-Polyadenylated RNA was extracted from the cells remaining in the syringe barrel after fine needle aspirate cytological diagnosis of 41 patients with breast lesions. Analysis of CD44 gene expression was performed by RT-PCR using primers flanking the site for insertion of the variant exons. The resulting products were separated on 1.2% agarose gels, transferred to nylon membranes using Southern blotting and hybridised with specific probes for standard (constitutive) and variant exons of this gene.Results-On hybridisation with the CD44 standard exon probe, the expected amplified product of approximately 482 base pairs was visualised in 22 of 41 samples examined. Further hybridisation with the "variant" exon probes (exons 7 (v2), 8 (v3), 9b (v4), 12 (v7), and 15 (v10)) on 12 of these samples showed the presence of large molecular variants in all of these samples. However, the expression pattern detected with the probes for exons 7 (v2), 8 (v3) and 9b (v4) differed among the patients.Conclusions-Expression of the standard and variant regions of the CD44 gene in cells remaining in the syringe after fine needle aspiration was demonstrated using RT-PCR. The 5' variant exon probes seemed to show different patterns of expression among the patients. Further studies are currently being conducted to determine whether there is any correlation between expression of the various components of this gene and cytological diagnosis. Using this method, it would be possible to study the expression of other candidate marker genes in breast cancer using fine needle aspirates.

Aims-Exon 7 of the human CD44 gene is overexpressed in many commonly occurring carcinomas. The aim of the study was to explore the diagnostic and therapeutic potential of this frequent abnormality.Methods-A new monoclonal antibody (mAb, M-23.6.1) and a polyclonal antibody (pAb,S-6127) to the corresponding antigen were raised by immunising mice and sheep, respectively, with a specially constructed fusion protein HIV2 (gp32)-CD44 exon 7.Results-Characterisation of mAb, M-23.6.1 by ELISA, western blotting, immunocytochemistry, and FACS analysis confirmed that it specifically recognises an epitope in the region between amino acids 19 and 33 of the peptide encoded by this exon. Western blotting experiments with two cell lines, RT112 and ZR75-1, known from RT-PCR data to be overtranscribing the exon, yielded a monospecific band of approximately 220 kDa, and immunocytochemistry showed discrete membrane staining on the same cell lines. Fluorescent antibody cell sorting (FACS) revealed binding to greater than 90% of the cells of each of these lines. Specificity of recognition of the antigen was shown by inhibition of the precise immunoreactivity typically seen in ELISA and Western blots, by pre-incubation with synthetic exon 7 peptide or fragments of it.Conclusions-The new antibodies will be useful tools for the further analysis of abnormal CD44 isoforms and their clinical implications.

Aims-To investigate the expression of CD44 proteins in exfoliated urothelial cells and in tumour tissues from bladder cancer patients. A further objective was to evaluate the diagnostic potential of the changes observed in the expression of these proteins as a marker for non-invasive detection of bladder cancer.Methods-Naturally voided urine specimens were collected from 47 patients with bladder cancer or severe urothelial dysplasia (n=3) and from a control group of 43 people with no evidence of neoplastic disease. Exfoliated urothelial cells floating in the urine were pelleted by centrifugation and lysed, and their constituent proteins extracted. The pattern of expression of CD44 proteins in each sample was examined by western blot analysis using a monoclonal antibody, Hermes 3, which recognises an epitope on the polypeptide backbone of the CD44 protein. Immunohistochemical studies were performed on neoplastic (n=10) and normal (n=4) bladder tissue specimens which were snap frozen in liquid nitrogen before examination with antibodies to CD44 gene products (CD44s and CD44v6).Results-Western blot analysis revealed several high molecular weight CD44 isoforms > 160 kDa in urine cell lysates from 75% of patients with histologically confirmed bladder cancer and in two of the three patients with severe dysplasia. Such patterns were not detected in the urine cell pellets from any persons in the control group. Immunohistochemical studies of the tissue distribution of CD44s and CD44v6 showed that the differentiation and maturation of the epithelial cells in the normal bladder mucosa is accompanied by a decrease in CD44 protein expression. However, carcinoma cells overexpress standard and variant CD44 isoforms and continue to do so as they proceed through the thickened epithelial layer to the luminal surface and after they are shed into the urine.Conclusions-The abnormal expression of CD44 proteins in exfoliated cancer cells may be a useful marker for the noninvasive diagnosis of bladder cancer.

Early detection of colorectal cancer (CRC) is desired for reducing its mortality rate. Recently, the feasibility of a new method for isolating colonocytes from feces was demonstrated, followed by direct sequencing analysis for detecting colorectal cancer. In the present study, gene expression analysis was conducted using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). One hundred and sixty-six patients with CRC and 134 healthy volunteers were enrolled. Messenger RNA expressions of CEA, MMP7, MYBL2, PTGS2 and TP53 in the colonocytes isolated from feces were analyzed by quantitative real-time RT-PCR. Beta-2-microglobulin, used for internal control, could not be detected in approximately 25% each of the CRC patients (39/166) and healthy volunteers (33/134). CEA expression did not differ significantly between CRC patients and healthy volunteers (P = 0.21). MMP7, MYBL2, PTGS2 and TP53 gene expressions were significantly higher in CRC patients than in healthy volunteers (P < 0.001). The overall sensitivity and specificity using these gene expressions were 58.3% (74/127, 95% CI; 49.2-67.0) and 88.1% (89/101, 95% CI; 80.2-93.7), respectively. The sensitivity was dependent on the tumor location (P = 0.01) and tumor size (P = 0.02), but not the tumor depth (P = 0.06) or cancer stage (P = 0.37). Gene expression analysis of colonocytes isolated from feces may be a useful method for CRC screening, if the number of isolated colonocytes is sufficiently high for analysis by quantitative real-time PCR. Therefore, improvement of the colonocyte retrieval system from feces may be necessary for the technique to be developed for clinical use.

Bladder cancer is among the top eight most frequent cancers. Its natural history is related to a combination of factors that impact on its aggressiveness. Cystoscopy and urine cytology are the currently used techniques for the diagnosis and surveillance of non-invasive bladder tumors. The sensitivity of urine cytology for diagnosis is not high, particularly in low-grade tumors. The combination of voided urine cytology and new diagnostic urine tests would be ideal for the diagnosis and follow-up of bladder cancer. However, in order to have some clinical utility, new diagnostic and/or prognostic markers should achieve better predictive capacity that the currently used diagnostic tools. None of the markers evaluated over the last years showed remarkable sensitivity or specificity for the identification of any of the diverse types of bladder cancer in clinical practice. The limitations of the known prognostic markers have led to the research of new molecular markers for early detection of bladder cancer. This research focused in particular on the discovery of biomarkers capable of reducing the need for periodic cystoscopies or, ideally, offering a non-invasive examination instead. In this review, we will examine various new markers of bladder cancer and their value in the diagnosis and follow-up of non-muscleinvasive bladder cancer. When compared with urine cytology, which showed the highest specificity, most of these markers demonstrated an increased sensitivity.

The diagnosis of both primary and recurrent bladder tumors currently relies upon the urine cytology and cystoscopy. Neither of these diagnostic tools is completely accurate. Prognostication of bladder cancer is largely based on pathologic tumor grade and stage. Over the past 2 decades, there is accumulating evidence that like many other cancers, bladder cancer, too, has a distinct molecular signature that separates it from other cancers and normal bladder tissue. Bladder tumors of different grades and stages even possess unique, and specific genotypic and phenotypic characteristics. Although recognition of several of these molecular alterations is possible by analyzing tumor tissue, urine, and serum samples, few if any of these "molecular markers" for bladder cancer are widely used in clinical practice. These markers include some that can be applied during the diagnostic work-up of symptoms (e.g., hematuria), those under surveillance for recurrence of superficial disease and forecasting long-term prognosis, or response to chemotherapy. In this review of molecular markers for bladder cancer, effectiveness of markers in each of these categories that are identifiable in the urine of patients with bladder cancer was examined. Many of the diagnostic markers appear to hold an advantage over urine cytology in terms of sensitivity, especially for the detection of low-grade superficial tumors. However, most markers tend to be less specific than cytology, yielding more false-positives. This result is more commonly observed in patients with concurrent bladder inflammation or other benign bladder conditions. Although there are several candidate markers for assessing prognosis or response to chemotherapy, studies of large patient populations are lacking. Further studies involving larger numbers of patients are required to determine their accuracy and widespread applicability in guiding treatment of bladder cancer.

The simultaneous or metachronous development of multifocal tumors with identical or variable histological features in the urothelial tract in a single patient is a well-known characteristic of urothelial cancer. To explain this phenomenon, two distinct concepts have been proposed: the 'field defect' hypothesis according to which urothelial cells in patients are primed to undergo transformation by previous carcinogenic insults and the 'single progenitor cell' hypothesis, which asserts that the multifocal development is caused by the seeding or intraepithelial spread of transformed cells. Results of recent molecular genetic studies support the 'single progenitor cell' hypothesis, and indicate that the genetic and phenotypic diversity observed in multifocal urothelial tumors is a consequence of clonal evolution from a single transformed cell. An understanding of the mechanism of the heterotopic recurrence of urothelial cancer may provide new prospects for early molecular detection and prevention of heterotopic recurrence of urothelial cancer.

The early detection of colorectal cancer is desired because this cancer can be cured surgically if diagnosed early. The purpose of the present study was to determine the feasibility of a new methodology for isolating colonocytes from naturally evacuated feces, followed by cytology or molecular biology of the colonocytes to detect colorectal cancer originating from any part of the colorectum.

Several simulation studies were conducted to establish the optimal methods for retrieving colonocytes from any portion of feces. Colonocytes exfoliated into feces, which had been retrieved from 116 patients with colorectal cancer and 83 healthy volunteers, were analyzed. Part of the exfoliated colonocytes was examined cytologically, whereas the remainder was subjected to DNA analysis. The extracted DNA was examined for mutations of the APC, K-ras, and p53 genes using direct sequence analysis and was also subjected to microsatellite instability (MSI) analysis.

In the DNA analysis, the overall sensitivity and specificity were 71% (82 of 116) of patients with colorectal cancer and 88% (73 of 83) of healthy volunteers. The sensitivity for Dukes A and B was 72% (44 of 61). Furthermore, the sensitivity for cancers on the right side of the colon was 57% (20 of 35). The detection rate for genetic alterations using our methodology was 86% (80 of 93) when the analysis was limited to cases in which genetic alterations were present in the cancer tissue.

We have developed a new methodology for isolating colonocytes from feces. The present study describes a promising procedure for future clinical evaluations and the early detection of colorectal cancers, including right-side colon cancer.

The natural history of non-muscle invasive bladder cancer is characterized by a high probability of recurrence and in the case of high-grade tumors, progression to muscle invasive cancer. This mandates a follow-up strategy designed to identify recurrences in the bladder early in their evolution in order to facilitate early intervention and ablation. Urine cytology is considered the gold standard urine biomarker. Although specificity exceeds 90% to 95%, its overall sensitivity ranges from 40% to 60% in expert hands and is both tumor grade and operator dependent. While cytology is an excellent test for detection of high-grade disease, the sensitivity is particularly weak for the detection of low grade tumors. This has spawned an entire field of research of in vitro diagnostic tests and cell-based assays in order to improve the diagnostic accuracy for detection of incident or recurrent disease. To date, the US Food and Drug Administration approved dipstick and immunoassays marketed as point-of-care tests. The point-of-care tests are intended for use as an adjunct to cystoscopy and cytology, and may have a role in the office evaluation of hematuria patients. Monoclonal antibody-based tests combined with cytology may improve the diagnostic accuracy and are superior to cytology alone. A recently approved cell-based assay, utilizing fluorescent in situ hybridization technology, may help resolve suspicious cytologies, and provide early and additional information about the biology of the bladder urothelium beyond that provided by cytology, a marker of disease relatively late in evolution. Novel promising markers are in various stages of clinical testing, and a panel of biomarkers may serve in the future as a feasible alternative to urine cytology and cystoscopy for the screening, detection, and follow-up of non-muscle invasive bladder cancer.

The polymerase chain reaction (PCR) is a highly sensitive and specific method for the detection of genetic material. Over the last few years, this method has been used increasingly for the molecular detection of disease. This report demonstrates the use of PCR in the diagnosis of bladder cancer. The principles of the method are shown by describing the detection of p53, CD44 and telomerase, as well as in microsatellite analysis.

Although the current system of classifying bladder cancer by stage and histological grade is very useful, it is still difficult to predict the natural progression of the disease either with or without therapy. Cystoscopy and urine cytology are currently the gold standards in the monitoring and diagnosis of bladder cancer. Classical urine cytology is, however, at least in the diagnosis of G1-tumors, characterized by a relatively low sensitivity. In the last few years, the molecular biological investigation of the basic mechanisms involved in carcinogenesis has provided a host of markers which are of potential diagnostic value for bladder cancer. We provide a current, comprehensive review of the literature on bladder tumor markers and summarize their diagnostic and prognostic potential. At present, no diagnostic marker with a comparable sensitivity and specificity to cystoscopy exists, given that cystoscopy has never been evaluated. The combined analysis of several tumor markers seems to be the most promising approach as an adjunct to cystoscopy. Moreover, the increasing simplification of test systems will increase their acceptance by clinicians.

The purpose of this study was to examine the occurrence of CD44 isoforms in breast carcinomas and their role in predicting clinical outcome.

Shock-frozen tumour tissues from 110 patients with breast carcinoma were examined by immunohistochemistry using antibodies directed against CD44s, v5, v6, v7 and v3-10. In addition, 80 of these tumours were available for quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CD44s and CD44v6. Immunohistochemically, the positive tumours showed cytoplasmic and/or membranous staining with all antibodies. Staining results did not correlate with histological subtype, lymph node status, status of steroid receptors, tumour size or age. Neither was any correlation found for overall and disease-free survival. Quantitative real-time RT-PCR of CD44s and CD44v6, however, revealed that expression of CD44v6 mRNA was significantly associated with lower pathological grade (Pearson chi(2) test P = 0.009; linear-by-linear association P = 0.003). Linear-by-linear association between CD44s mRNA expression and lower pathological grade was also seen (P = 0.02). Survival analysis with the Kaplan-Meier method demonstrated that increased CD44s mRNA expression was significantly associated with both disease-free survival and overall survival (P = 0.0185 and P = 0.0344, respectively). A similar trend for CD44v6 mRNA expression was seen in these cases, but the difference was not significant.

Quantitative real-time RT-PCR revealed clinical correlations of CD44s and CD44v6 mRNA expression in breast carcinomas while immunohistochemistry for the protein expression of CD44s and other CD44 variants did not. This contradictory result merits further studies concerning the clinical impact of CD44 molecules in breast carcinomas.

Splice variants of the cell surface glycoprotein CD44 have been reported to be associated with the progression of various human tumors. The aim of this study is to determine the correlation between the expression of CD44 isoforms, especially CD44 variant 2 (CD44v2), and the clinicopathological features of head and neck squamous cell carcinomas (HNSCCs). The expression of CD44 isoforms was evaluated immunohistochemically in paraffin-embedded tissues from 89 primary lesions, using monoclonal antibodies against CD44 standard (CD44st), CD44 variant 6 (CD44v6) and CD44v2. Cancer tissues from 89 (100%), 85 (95.5%) and 59 (66.3%) patients showed positive immunoreactivity for CD44st, CD44v6 and CD44v2, respectively. A significant correlation was observed between the down-regulation of CD44v2 and poorer differentiation of the tumor cells (P = 0.02). We could not find any significant correlation between the expression of CD44v2 and T stage or N stage (lymph node status). However, the rate of positive cervical lymph node metastasis tended to increase with reduced expression of CD44v2 (P = 0.08). Down-regulation of CD44v2 expression was correlated with shorter overall survival (P = 0.01). Furthermore, Cox's multivariate analysis revealed that only CD44v2 expression and lymph node status were independent prognostic factors. These findings suggest that down-regulation of CD44v2 expression may be one of the biological markers for the degree of malignancy in HNSCCs.

The CD44 variant (CD44v) isoforms have been noted as markers for tumour metastasis and prognosis in several adenocarcinomas.

To investigate whether CD44v, especially the CD44v2 (v2) isoform, may be a useful prognostic factor for patients with oesophageal squamous cell carcinoma, using a recently developed monoclonal antibody against a v2 epitope.

233 patients (211 men and 22 women; mean age 61.9 years), with oesophageal squamous cell carcinomas curatively removed without additional treatment between 1987 and 1996 at the National Cancer Center Hospital, were analysed for CD44v expression.

The expression of CD44v was evaluated immunohistochemically using monoclonal antibodies against epitopes of the standard and variant protein, in paraffin embedded oesophageal squamous cell carcinoma tissue from 233 patients who had undergone cervical, mediastinal, and abdominal lymphadenectomy (three field dissection) for oesophagectomy. The data were evaluated for any correlation with clinicopathological indices or prognosis.

Although total CD44 and CD44v6 (v6) were respectively observed in 99% and 97% of the cancer specimens, the expression of v2 was only 30%. Patients whose tumours were v2 positive had a significantly better prognosis than those whose tumours were v2 negative (p = 0.031). Furthermore, in patients without lymph node metastasis, v2 positivity alone was a significant independent factor of prognosis (relative risk of death associated with v2 negativity, 4.7; p = 0.037) in multivariate analysis.

These results indicate that v2 is a useful marker for clinical prognosis in patients with oesophageal squamous cell carcinoma. Particularly in patients without lymph node metastasis, v2 status may thus have implications for the use of adjuvant chemotherapy and/or radiotherapy in patients with oesophageal cancer at an early stage.

The majority of papillary transitional cell carcinomas of the bladder are localized tumors at initial diagnosis; identification of those developing recurrence and an aggressive behavior is important. CD44 variant proteins have been implicated in tumor progression and metastasis, and a correlation with adverse prognosis has been demonstrated in a variety of human tumors. Here, the usefulness of conventional CD44 protein immunohistochemistry as a prognostic parameter for recurrence of superficial transitional cell carcinomas was assessed in paraffin sections of 241 tumors with long-term follow-up. A highly significant association was found between focal loss of CD44v3 and -v6 immunostaining and short recurrence-free interval in noninvasive (pTa) transitional cell carcinomas (P = 0.005), but not in minimally invasive (pT1) carcinomas (P = 0.78). Our results indicate the value of conventional CD44 immunohistochemistry as an additional tool for identifying patients at high risk for recurrence of pTa transitional cell carcinomas. They also point to biological differences between noninvasive and minimally invasive transitional cell carcinomas of the bladder.

Receptor for hyaluronan (HA)-mediated motility (RHAMM) is a receptor for HA-mediated motility and its expression is correlated with malignancy of ras-transformed cells in that binding of HA to this receptor activates their migratory ability. CD44, a cell surface receptor for HA is also implicated in metastatic behavior of some cancer cells. In this study we examined the relationships of cancer progression with mRNA levels of RHAMM, CD44 (all forms), and exon 6 of CD44 using the real-time reverse transcriptase-polymerase chain reaction method in specimens of colon cancers at different diagnostic stages from 30 patients. Increased mRNA levels of RHAMM were observed in 29 specimens (97%), CD44s (all forms) in 21 specimens (70%), and its exon 6 in 19 specimens (63%) in comparison with those in the corresponding noncancerous tissue specimens. A statistically significant correlation between RHAMM expression and cancerous specimens at any of Dukes' stages A, B, and C was found, and the overexpression of CD44 mRNAs was confirmed in specimens at Dukes' stage C. Thus, our present study for the first time suggests that RHAMM expression may be a clinically useful indicator of colon cancer.

Recent research has revealed the existence of specific mutations in cancer. These mutations are being investigated as targets to find subjects at high risk for cancer, to detect early cancer, to detect the early recurrence of established cancer, and to find micrometastasis. These mutations are reviewed for the major anatomic sites. Some of the clinical issues related to the application of these mutations and the limitations of using molecular targets are also considered. Current methods for determining the risk of cancer are reviewed. Risk assessment is essential for defining cohorts for chemoprevention and other interventions. The concept of using surrogate anatomic and functional sites for estimating risk is introduced. Finally, the increasing complexity of molecular genetic analysis and the biologic heterogeneity of cancer are discussed in relation to early detection.

The CD44 proteins form a ubiquitously expressed family of cell surface adhesion molecules involved in cell-cell and cell-matrix interactions. The multiple protein isoforms are encoded by a single gene by alternative splicing and are further modified by a range of post-translational modifications. CD44 proteins are single chain molecules comprising an N-terminal extracellular domain, a membrane proximal region, a transmembrane domain, and a cytoplasmic tail. The CD44 gene has only been detected in higher organisms and the amino acid sequence of most of the molecule is highly conserved between mammalian species. The principal ligand of CD44 is hyaluronic acid, an integral component of the extracellular matrix. Other CD44 ligands include osteopontin, serglycin, collagens, fibronectin, and laminin. The major physiological role of CD44 is to maintain organ and tissue structure via cell-cell and cell-matrix adhesion, but certain variant isoforms can also mediate lymphocyte activation and homing, and the presentation of chemical factors and hormones. Increased interest has been directed at the characterisation of this molecule since it was observed that expression of multiple CD44 isoforms is greatly upregulated in neoplasia. CD44, particularly its variants, may be useful as a diagnostic or prognostic marker of malignancy and, in at least some human cancers, it may be a potential target for cancer therapy. This review describes the structure of the CD44 gene and discusses some of its roles in physiological and pathological processes.

CD44 is a family of cell surface transmembrane glycoproteins members which differ in the extracellular part by sequences derived by alternative splicing of 10 variant exons (v1-v10). CD44 proteins containing such variant sequences have been implicated in tumor metastasis formation. Here, we have evaluated the expression of CD44 variants by immuno-histochemistry in primary breast cancer samples of 237 node-negative and 230 node-positive patients. For the analysis of samples derived from node-negative patients, the exon-specific antibodies used were DIII, vff7 and vff18 (v6), vff17 (v7/v8), fw11.24 (v9) and vff16 (v10). With the different antibodies which recognize v6 epitopes, the majority of tumors were positively stained (> or = 65% of the tumors) with varying intensities. Thirty-nine percent of the tumors were positively stained with the antibody vff16, and approximately half of the tumors with the antibodies vff17 and fw11.24. The expression of CD44 v6 epitopes in tumors from node-negative patients was associated with a favorable prognosis, both upon univariate and multivariate analysis. The expression of CD44 v7/8, v9 or v10 epitopes was not significantly related with relapse-free survival. Samples from node-positive patients were only examined with the antibodies vff7, vff17 and vff18. The staining with none of these antibodies was correlated with the length of relapse-free survival of the patients. Our data suggest that, generally, the usefulness of knowledge of CD44 variant expression is of limited value for assessing the risk of relapse in patients with primary breast cancer. However, the expression of exon v6 of CD44 may be a marker to identify patients with a relatively favorable prognosis in node-negative patients.

The expression pattern of CD44 standard and variant isoforms are prognostically significant in a number of malignancies. The aim of this study was to evaluate the role of the standard isoform of CD44 in predicting the clinical behaviour of rhabdomyosarcoma. Immunohistochemical analysis of CD44 was undertaken using a panel of antibodies recognizing the three core domains of the CD44 molecule. Labelling was repeated in triplicate and reported blind with respect to histological type and outcome. Tumours were characterized as positive in more than 60% of tumour cells labelled and negative if less than 40% of tumour cells labelled. Tumours with 40-60% of tumour cells labelling were considered indeterminate. Eleven of 20 favourable histology tumours were positive for CD44 compared with one of seven unfavourable tumours (P = 0.07). Eleven of 12 patients with CD44-positive tumours are alive in first remission compared with five of 15 CD44-negative tumours (P = 0.001). Expression of CD44 correlates directly with prognosis; however, larger studies are required so that multivariate analysis can be undertaken.

CD44 is a widely expressed cell surface adhesion molecule in which various isoforms arise from alternative RNA splicing mechanism. Overexpression of specific CD44 splice variant, i.e., CD44v8-10, has been found in several human malignancies and is considered to be associated with tumor progression and metastasis. We have demonstrated a novel molecular approach, CD44v8-10/CD44v10 competitive reverse transcription-polymerase chain reaction (CC-RT-PCR) for the detection of cancer cells overexpressing CD44v8-10 by the measurement of the transcriptional level of CD44v8-10 relative to that of CD44v10 (v8-10/v10 ratio). In this study, we initially examined the expression of CD44 splice variants in human urothelial cancers and their adjacent normal urinary tissues by RT-PCR. Any CD44 variant isoforms were barely detectable in normal urinary tissues, whereas CD44v8-10 was predominantly expressed in 23 of the 30 (77%) urothelial cancer specimens. We then applied CC-RT-PCR to spontaneously voided urine samples from patients with 80 urothelial cancer and 50 various benign urologic diseases. The CC-RT-PCR analysis revealed that all of the samples associated with benign diseases presented a predominant expression of the CD44v10 transcript (the v8-10/v10 ratios = 0.00-0.87), whereas 62 of the 80 samples associated with urothelial cancers mostly expressed the CD44v8-10 transcript (the v8-10/v10 ratios > 1.00). In addition, the positivity rate obtained by the CC-RT-PCR analysis was high regardless of the pathological grade of the urothelial cancers, although the sensitivity of the cytological examination declined with decreasing tumor grade. Our findings suggest strongly that CC-RT-PCR is a non-invasive useful tool for the diagnosis of urine samples from patients with urothelial cancer.

To investigate the expression of CD44 isoforms containing variant exon 6 (v6) in a well characterised cohort of patients with non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukaemia (CLL), and to correlate this with phenotype and disease course.

Cryostat sections of OCT embedded diagnostic nodal material from NHL patients and cryopreserved mononuclear preparations from CLL patients were used as sources of RNA. After reverse transcription, PCR was carried out with amplimers positioned at either side of the variant exon insertion site to amplify all possible CD44 isoforms. Those isoforms containing v6 were identified after Southern blotting and hybridisation with a radiolabelled oligonucleotide.

Of 32 NHL samples analysed, 16 did not express CD44 isoforms containing v6, six expressed an isoform containing exon v6 alone, and 10 expressed v6 long isoforms which contained exon v6 in addition to other variant exons. These data did not correlate with lymphoma classification, disease staging, or the presence or absence of extranodal disease. However, those patients expressing v6 long CD44 isoforms had a worse overall survival than those that did not. The plateau of the survival curves was 50% compared with 82%. No v6 long isoforms were detected in the 21 CLL samples investigated.

The expression of v6 long CD44 isoforms is associated with aggressive disease in NHL, independent of grade, stage, or presence of extranodal disease.

Markedly increased overall levels of CD44 transcripts and proteins have been recognized in many tumors and the inappropriate expression and abnormal assembly of the CD44 variable exons has been linked to both tumor growth and metastatic potential. We have also previously observed the aberrant inclusion of intron 9 in CD44 mRNA transcripts in tumor tissues. In this study we assessed whether such retention is specific to certain introns or is a more general phenomenon affecting CD44 gene expression in tumor cells. Intron 18 was cloned and sequenced from genomic DNA and the novel sequences analyzed and used to create intron 18-specific probes. The newly characterized intron was found to have consensus 5' splice site and branchpoint sequences but a suboptimal 3' splice site. The status of CD44 intron 18 retention or excision was assessed in a colon tumor cell line (HT29) and in tissue from 20 colorectal tumors and matched normal mucosa. The intron was shown to be retained in transcripts from 15 of the 20 (75%) carcinomas but in only 3 of the 20 (15%) matched normal samples. These results compare with 80% retention of CD44 intron 9 in colonic carcinoma tissue mRNA and confirm that multiple abnormalities of CD44 mRNA processing occur in tumor cells.

Since the CD44 variant 6(v6) molecule has been noted as a marker for tumor metastasis and prognosis in several tumors, we examined whether or not v6 is a useful marker for evaluating the prognosis of pancreatic cancer patients. In addition, we attempted to assess the clinicopathological implications for pancreatic cancer of the variant 2 (v2) isoform using a recently developed monoclonal antibody against a v2 epitope. The expression of CD44 variants was evaluated immunohistochemically in paraffin-embedded pancreatic cancer tissues from 42 patients who were confirmed surgically and histologically to have received curative resection. An indirect immunoperoxidase method was used with monoclonal antibodies against epitopes of the standard (CD44s) portion, v6 and v2. Protein expression data were evaluated statistically for any correlations with the length of survival or with histological parameters. The expression of CD44v6 and v2 was observed only in tumor cells, if at all. On the other hand, expression of total CD44 (including CD44v, as well as CD44s) was observed in both tumors and adjacent normal sites. Tumor tissue from 21 (50%) and 16 (38%) patients showed positive immunoreactivity with mAb 2F10 (anti-CD44v6) and mAb M23.6.1 (anti-CD44v2), respectively. The expression of CD44v6 and v2 was correlated with decreased overall survival (P = 0.0160 and P = 0.0125, respectively). A significant correlation was obtained between CD44v2 peptide expression and vessel invasion (P = 0.026). These results suggest that CD44v2 and CD44v6 may be useful markers for poor prognosis in curatively resected primary pancreatic cancer.

CD44v6 expression appears to be associated with adverse prognosis and propensity for metastasis in patients with colorectal cancer. However, expression of CD44 variants in different tumour stages has been poorly characterised. CD44 variant expression was investigated in normal colonic mucosa (n = 36), colorectal adenomas (n = 15), carcinomas (n = 62) and metastases (n = 6) by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting with exon-specific probes. High frequencies of CD44 standard (CD44s) and CD44 epithelial (CD44e) were observed in normal and neoplastic tissue. CD44v2 was seen predominantly in adenomas (27%) and UICCI carcinomas (29%). CD44v5 expression was low in normal mucosa (3%), higher in adenomas and carcinomas (29-33%), independent of tumour stage. CD44v6 expression was low in normal mucosa (6%) and higher in adenomas (47%) and carcinomas (42%). Surprisingly, a significant decrease of CD44v6 was observed in metastatic primary tumours (8%) and metastases (17%) (UICCIV) (P < or = 0.05). Therefore, the concept of CD44v6 conferring metastatic potential to malignant cells cannot be supported by our data.

The cell adhesion molecules are ubiquitous recognition molecules that allow cells to communicate with one another and their environment. Through these molecules, complex alterations in the cytoplasmic messenger pathways and the microfilamentous cytoskeleton can lead to profound alterations in cell division, differentiation, behaviour, and function (fig 9). It is difficult to conceive of a group of molecules that could be more important to pathologists and to their understanding of disease processes.

Recent investigations have shown that CD44 variant exons are frequently overexpressed in human colorectal adenocarcinoma. The aim of this study was to investigate abnormal expression of the CD44 gene in exfoliated cells from patients with colorectal cancer.

Exfoliated cells in feces from 25 patients with colorectal cancer before and after surgery and from 15 healthy volunteers were analyzed. CD44 standard, variant 6, and variant 10 messenger RNA (mRNA) expressions were examined in the exfoliated cells in feces by using reverse-transcription polymerase chain reaction followed by Southern hybridization with exon-specific probes.

CD44 standard mRNA was detected in all samples before and after surgery and in all healthy volunteers. CD44 variant 6 and variant 10 mRNA were detected in 17 of 25 patients (68%) and 15 of 25 patients (60%), respectively, in individual feces obtained before surgery. CD44 variant 6 mRNA and variant 10 mRNA were detected in postoperative samples in 3 of 25 patients (12%) and 7 of 25 patients (28%), respectively. Fifteen of 17 patients who were positive for CD44v6 based on preoperative fecal samples became negative after surgery (88.2%). Similarly, 12 of 15 patients who were CD44v10 positive in preoperative fecal samples were negative postoperatively (80%).

These results suggest that analysis of CD44 variant expression in the exfoliated cells in feces can provide a noninvasive diagnostic test for colorectal cancer.

We have established a novel quantitative method based on the allele-specific PCR, which uses the linearly amplified fragment of the PCR products as the internal control. The improved characteristics of the procedure are the high sensitivity for quantitation of the mutant alleles at ratios of up to 1:10000 and the reduced necessity of the optimization of the PCR conditions for each mutation. Using this modified allele-specific PCR, we could quantify the tumor alleles in the urine sediments of three patients with urothelial cancers that harbored different p53 gene mutations. This method can be applied to other genetic targets that have other types of alterations, such as deletions or insertions.

Small cell carcinoma of the urinary bladder has a more aggressive biological potential than urothelial carcinoma, but its morphology may overlap with poorly differentiated urothelial carcinoma. The CD44 family of glycoproteins mediates cell-cell and cell-matrix adhesion. Aberrant regulation of expression of CD44, and particularly its v6 variant exon, has been correlated with aggressive or metastatic phenotype of some cancers. We measured the degree of protein expression of CD44v6 in small cell and urothelial bladder carcinoma.

Immunohistochemical staining was performed with monoclonal antibodies against CD44v6. Immunoreactivity was absent in 25 of 27 cases of small cell carcinoma, with two showing only weak staining of fewer than 10% of cells. In contrast, all 12 cases of moderately or poorly differentiated urothelial carcinoma displayed moderately intense reactivity in 50% to 100% of cells.

CD44v6 immunostaining discriminates cases of poorly differentiated urothelial carcinoma from small cell carcinoma. It also highlights the presence of mixed small cell urothelial differentiation when present.

In order to clarify the relation between expression of individual CD44 variant exons and tumor progression, 34 endometrial carcinomas (endometrioid type) were investigated, as well as 27 samples of normal endometrium, using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization (SBH). Western blotting was also performed for comparison of protein levels with the results of the RT-PCR/SBH methods. Analysis of gross CD44 splicing patterns demonstrated high-level expression of variant isoforms in endometrial carcinomas as compared with normal endometrium. Exon-specific RT-PCR/SBH assays revealed large, abundant transcripts of individual variant exons in particular v3, v4, and v5, in tumors, but these isoforms were also expressed in normal endometria, suggesting a lack of tumor-specificity. No individual CD44 variant transcripts were associated with any of the prognostic factors investigated. Parallel observations showed variant CD44 transcripts to be more readily detectable than protein isoforms in the same samples. These findings indicate that in endometrial carcinomas, expression of individual variant CD44 exons is markedly up-regulated, but this molecule may not be useful as a consistent indicator of tumor progression.

We examined the expression of CD44 isoforms in samples of breast cancer tissues from 95 patients by reverse transcription-polymerase chain reaction and immunohistochemistry, and tried to correlate the results with survival period. At the RNA level, expression of exon v2 was observed in 33 (35%) and that of v6 in 69 (73%) of the 95 specimens. Patients with CD44v2 mRNA expression had significantly shorter survival times than those with v2-negative tumors (P = 0.05), but there was only a weak correlation, if any, between v6 mRNA expression and overall survival (P = 0.06). Tumor tissue from 22 (23%) and 72 (76%) patients showed positive immunoreactivity with monoclonal antibody (mAb) M23.6.1. (CD44v2) and mAb 2F10 (CD44v6), respectively. Immunohistochemical evidence of CD44v2 peptide expression correlated with overall survival (P = 0.02), but there was no such association with CD44v6 expression in these tumors (P = 0.67). There were significant correlations between v2 immunoreactivity and higher histological grade and lower levels of estrogen and progesterone receptor. There was no significant correlation between v6 immunoreactivity and such clinicopathological characteristics. Although the expression of v2 was significantly associated with reduced overall survival, it was not an independent prognostic factor because it also correlated with progesterone receptor status. These findings suggest that v2 isoform expression might have more value than v6 expression for clinical use.

CD44 is a cell surface glycoprotein implicated in such diverse biologic processes as lymphocyte activation and homing, extracellular matrix adhesion, and cellular migration. Primary transcripts of the CD44 gene can be alternatively spliced to produce a variety of messenger RNA (mRNA) species. The standard form of CD44 mRNA contains sequences from at least 20 genomic exons; variant mRNAs contain sequences from one or more additional exons (v1-10). Predominant expression of a specific CD44 variant, i.e., CD44v8-10, in several human carcinomas has been described previously. In this study, we developed a novel molecular approach for detecting cancer cells that overexpress CD44v8-10 mRNA.

After finding that CD44v8-10 was predominantly expressed in non-small-cell lung and bladder carcinomas and that CD44v10 was predominantly expressed in leukocytes, we developed a competitive reverse transcription-polymerase chain reaction assay (CC-RT-PCR) that allows quantification of the relative expression of these two mRNA species in clinical specimens (i.e., determination of a v8-10/v10 ratio). CC-RT-PCR analysis was applied to pleural effusion specimens from patients with benign or malignant lung diseases as well as to spontaneously voided urine samples from patients with benign or malignant urologic diseases.

Fifty two of 54 samples from patients with benign diseases expressed CD44v10 predominantly (v8-10/v10 ratio < or = 0.65), whereas 46 of 61 samples from patients with malignant diseases expressed CD44v8-10 predominantly (v8-10/v10 ratio > 1.00) (two-sided P < .001). CC-RT-PCR detected predominant expression of CD44v8-10 in cytologically negative samples from 11 patients who were later diagnosed with malignant disease.

CC-RT-PCR analysis of CD44v8-10 expression could be an important adjunct to cytologic examination in cancer diagnosis, especially in detecting exfoliated cancer cells in pleural effusions and urine.

Altered expression of CD44 has been implicated in tumor progression and metastasis in multiple neoplasms.

CD44 expression in archival tissues of prostate carcinoma was examined by immunohistochemistry with monoclonal antibodies against core CD44 and the RNA splice variant CD44v6 (v6).

Core CD44 expression was reduced in the majority of primary neoplastic foci (n = 94) and loss of expression correlated with increasing Gleason grade. Staining for v6 was absent in most carcinomas and metastases. Expression of core CD44 in pelvic lymph node (n = 27) and bone metastases (n = 21) was significantly reduced. In addition, CD44 expression correlated with cytoarchitecture. Tall columnar tumor cells typically stained positively, yet more rounded cells forming cribiform structures or nests showed reduced expression. All cases of high-grade prostatic intraepithelial neoplasia were positive for core CD44 yet, there was decreased expression in cribiform and micropapillary variants.

The majority of clinically relevant human prostatic carcinomas and metastases downregulate expression of CD44. Additional studies to determine whether CD44 cell surface expression relates to clinical outcome independent of other established clinicopathologic risk factors are warranted.

CD44 is a major cell surface receptor for the glycosaminoglycan, hyaluronan (HA). CD44 binds HA specifically, although certain chondroitin-sulfate containing proteoglycans may also be recognized. CD44 binding of HA is regulated by the cells in which it is expressed. Thus, CD44 expression alone does not correlate with HA binding activity. CD44 is subject to a wide array of post-translational carbohydrate modifications, including N-linked, O-linked and glycosaminoglycan side chain additions. These modifications, which differ in different cell types and cell activation states, can have profound effects on HA binding function and are the main mechanism of regulating CD44 function that has been described to date. Some glycosaminoglycan modifications also affect ligand binding specificity, allowing CD44 to interact with proteins of the extracellular matrix, such as fibronectin and collagen, and to sequester heparin binding growth factors. It is not yet established whether the HA binding function of CD44 is responsible for its proposed involvement in inflammation. It has been shown, however, that CD44/HA interactions can mediate leukocyte rolling on endothelial and tissue substrates and that CD44-mediated recognition of HA can contribute to leukocyte activation. Changes in CD44 expression (mainly up-regulation, occasionally down-regulation, and frequently alteration in the pattern of isoforms expressed) are associated with a wide variety of cancers and the degree to which they spread; however, in other cancers, the CD44 pattern remains unchanged. Increased expression of CD44 is associated with increased binding to HA and increased metastatic potential in some experimental tumor systems; however, in other systems increased HA binding and metastatic potential are not correlated. CD44 may contribute to malignancy through changes in the regulation of HA recognition, the recognition of new ligands and/or other new biological functions of CD44 that remain to be discovered.

The CD44 gene codes for a family of heavily glycosylated cell surface proteins that have been linked with tumour metastasis. The aim of the study was to analyse the expression of CD44 messenger RNA in colorectal cancer.

The expression of CD44 variants 2 and 7 in colorectal tumour samples was compared with that in normal colon and lymphocytes from 59 patients using reverse transcription-polymerase chain reaction followed by blot hybridization with exon-specific probes, and a nested polymerase chain reaction.

All samples of tumour and metastatic tissue showed complex overexpression of many alternatively spliced products of the CD44 gene. Normal colon, liver and lymphocytes predominantly expressed the standard form of the CD44 molecule (CD44S) with low levels of two or three variants hybridizing to exons v2 and v7.

Deranged CD44 gene activity in colorectal cancer cells is confirmed. The analysis of CD44 gene expression may provide a promising marker for the early detection of colonic tumours.

Cancer cells release various antigens, some of which appear in the urine. Oral autourotherapy is suggested as a new treatment modality for cancer patients. It will provide the intestinal lymphatic system with the many tumor antigens against which antibodies may be produced. These antibodies may be pierced through the blood stream and attack the tumor and its cells.