CD44 Transcripts Research Articles

CD44 expression in benign, premalignant, and malignant ovarian neoplasms: relation to tumour development and progression

To clarify the possible role of CD44 expression in ovarian tumour development and progression, an immunohistochemical investigation was undertaken of a series of 115 carcinomas, 32 tumours with low malignant potential (LMP), and 53 cystadenomas. A combination of the reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization (SBH) assays was also performed for 17 malignant, four LMP, six cystadenoma, and seven normal ovarian samples. Immunoreactivity scores for CD44s, CD44v3, and CD44v6 were significantly higher in LMP and malignant tumours than in the benign or normal cases, in line with the results of gross mRNA-based assays. Exon-specific RT-PCR/SBH assays revealed that the expression of large CD44 transcripts containing v6 to v8 exons and small isoforms containing v2 and v3 was common among normal and neoplastic tissues, while a simultaneous increase of large isoforms containing v2 to v5 was also revealed in LMP and malignant tumours. In ovarian carcinomas, the scores for CD44s, CD44v3, and CD44v6 were inversely related to the FIGO stage, but there was no association with lymph node status or expression of hormone receptors. Multivariate analysis revealed loss of CD44v3 expression to be an independent factor for poor survival. The findings indicate that CD44 is up-regulated during the development of ovarian carcinomas but is subsequently down-regulated during their progression, resulting in aggressive behaviour and an unfavourable prognosis. Copyright © 1999 John Wiley & Sons, Ltd.

CD44 isoforms distinguish between bone marrow plasma cells from normal individuals and patients with multiple myeloma at different stages of disease.

CD44 variant isoforms (CD44v) have been shown to be important factors in adverse prognosis in hematological malignancies. To investigate whether CD44 expression is associated with malignant transformation in multiple myeloma, RNA and protein expression of CD44 standard (CD44s) and CD44v4, v6, v9, v10 containing isoforms was compared on bone marrow plasma cells from normal individuals and myeloma patients at different stages of disease. CD44s protein expression is strongly decreased on myeloma plasma cells and non-malignant B cells in affected bone marrow of myeloma patients, while no differences in CD44s expression were found between blood B cells from normal individuals and myeloma patients. CD44v isoforms were expressed on plasma cells in the majority of normal and myeloma samples analyzed. CD44v9 and v10 containing isoforms were differentially expressed on bone marrow plasma cells from normal individuals (predominantly CD44v9+v10+) and myeloma patients with stable (predominantly CD44v9-v10+) or progressive (predominantly CD44v9+v10- disease. Normal and myeloma plasma cells contained CD44 mRNA transcripts consisting of multiple CD44v exons. In addition, CD44v9 positive myeloma cells carried large CD44 transcripts. These results imply that detection of CD44v isoforms may be a valuable diagnostic tool for monitoring myeloma disease progression and response to treatment.

Multiple Intron Retention Occurs in Tumor Cell CD44 mRNA Processing

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.

Up-regulation of CD44 variant exon expression in endometrial carcinomas: analysis of mRNA and protein isoforms, and relation to clinicopathological factors.

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.

Cellular and molecular pathology of gastric carcinoma and precursor lesions: A critical review.

The cellular and molecular pathology of gastric cancer and its precursors are reviewed and discussed. Gastric carcinogenesis is a multistep phenomenon, beginning with precancerous conditions. Among these, adenoma is a direct precursor, because of the dysplastic nature of its cells. However, gastric adenoma is relatively rare. Chronic atrophic gastritis (CAG) is the most common precancerous condition, in which intestinal metaplasia often occurs. Carcinoma develops in CAG through stages of hyperplasia and dysplasia involving both metaplastic and non-metaplastic glands. Molecular alterations, including replication error and p53 and APC gene mutation and aneuploidy have been found in some of these conditions, confirming their role in carcinogenesis. Carcinomas of the stomach are heterogeneous in cellular composition. Both intestinal and gastric types of cells are found in all types of tumors, indicating the unique characteristics of gastric cancer. Many molecular lesions have been found in gastric carcinomas. Basic changes involve replication errors, telomerase activity, and aberrant CD44 transcripts. Many other changes often show differences in the frequency of their occurrence between the two major histological types of gastric carcinoma: well differentiated versus poorly differentiated, or intestinal type versus diffuse type. The timing and frequency of these changes in the stomach differ from the timing and frequency in colonic carcinogeneis. Pathological evaluation remains reliable and meaningful, in basic research as well as clinical management. To obtain correlation with molecular alterations, the need for detailed pathologic classification of gastric carcinoma is recognized, taking into account its biologic behavior and grades of cell differentiation.The cellular and molecular pathology of gastric cancer and its precursors are reviewed and discussed. Gastric carcinomas are unique in their heterogeneity in both cellular composition and molecular changes.

Clinical implications of anomalous CD44 gene expression in neoplasia.

An intensive search continues for reliable markers that would be clinically useful in the diagnosis of small tumors and in the evaluation of their predicted clinical outcome. One potential marker, extensively studied in human samples is the cell surface adhesion molecule CD44. The single CD44 gene codes for a large family of cell surface proteins by alternative splicing and severe abnormalities have been observed in the patterns of its expression in many types of human tumors using both protein and RNA-based analyses. These abnormalities are manifested by markedly increased levels of unusual transcripts and proteins in tumor cells compared to the corresponding normal tissues. Aberrant processing of immature CD44 transcripts has also been observed in tumor cells and this can lead to the inappropriate retention of introns and to the use of cryptic splice sites in the mRNA. Inappropriate expression patterns of the alternatively spliced exons have also been linked both to tumor progression and to metastatic potential. The clinical relevance of all these observations is demonstrated by the frequent detection of these abnormalities in samples from malignant tumors of many different organs and by their presence in pre-invasive and high risk pre-cancerous lesions. This article reviews the current information regarding the expression of the CD44 gene in tumor cells and its potential use as a marker in clinical evaluation. The overall conclusion is that with the use of the latest assay techniques and perhaps in combination with other molecular markers, analysis of CD44 expression can provide new and powerful assays for the detection of neoplastic disease.

The link between integration and expression of human papillomavirus type 16 genomes and cellular changes in the evolution of cervical intraepithelial neoplastic lesions.

We have matched a PCR assay which detects disruptions in the E2 reading frame of human papillomavirus type 16, with RNA in situ hybridization patterns and shown that in 15 out of 16 cervical intraepithelial neoplastic (CIN) III lesions and in 19 out of 19 tumours, the E2 gene is disrupted with no detectable E2 transcripts. Varying levels of E6-E7 transcripts are detected in CIN III lesions, with stronger signals in tumours. The cytokeratin profile of most tumours: cytokeratin 10-, 14- and 19-positive and 4-, 13- and 18-negative, is also detected in CIN III lesions. The changes in levels of alpha 2, beta 1 and beta 4 integrins, CD44 and E-cadherin occur during the evolution of high-grade CIN lesions. Increases in the levels of expression of CD44 and E6-E7 transcripts, coupled with changes in the cellular localization of the Notch protein, define the transition from CIN III lesions to tumours.

Changes in CD44 Expression during Carcinogenesis of the Mouse Colon

CD44 glycoprotein is the main extracellular receptor for hyaluronic acid. The CD44 gene is composed of 20 exons and encodes a variety of isoforms generated by alternative splicing of 10 variant exons. Overexpression of discrete CD44 isoforms containing products of variant exons have been implicated in the progression of cancer, including human colon carcinoma. The pattern of CD44 transcripts changes during early colorectal carcinogenesis, and their relation to CD44 protein expression remains to be defined under experimental conditions. In the current study we investigated CD44 expression in a murine model of human colon adenoma/carcinoma. Colon tumors were induced in 19 ICR/Ha mice by 1,2-dimethylhydrazine injections and CD44 expression was studied by RT–PCR/Southern blot analysis as well as immunohistochemistry. CD44 transcripts were strongly overexpressed in tumors compared to normal colon. Both neoplastic and normal colon samples exhibited the same species of CD44 transcript representing standard and variant isoforms. Seventy-five percent of neoplasms contained foci of CD44-positive tumor cells, whereas in normal colon the epithelial immunoreactivity was confined to the crypt base. Immunostaining of neoplastic cells was heterogeneous and there was a significant tendency toward the progressive loss of CD44 immunoreactivity in large invading tumors. It is concluded that early events in murine colorectal carcinogenesis are characterized by a marked global overexpression of standard and variant CD44 transcripts.

Molecular genetic characterization of alternatively spliced CD44 transcripts in human stomach carcinoma.

CD44 is a member of cell surface glycoproteins which are involved in cell-matrix adhesion and tumor metastasis. Certain types of tumors express complex CD44 isoforms generated by alternative splicing of 2v-10v exons, and their expression appears to promote metastasis of tumor cells. Using a nested RT-PCR, we analyzed expression of CD44 variants in 26 stomach carcinoma, 21 matched normal tissues, and 2 carcinoma cell lines. We observed frequent and complex patterns of CD44 variant expression in tumor tissues. While exons 6v and 7v expression was detected in most normal and tumor tissues, exon 9v was most rarely detected. Exon 5v showed a significantly frequent expression in carcinoma, suggesting that its expression might contribute to the malignant progression. While exon 9v was frequently observed in diffuse-type tumors, the other 8 variant exons including 6v showed more frequent expression in intestinal-type tumors. Exons 9v and 10v were predominantly expressed in advanced tumor tissues and exon 8v was expressed more frequently in tumors of lymph node metastasis. We believe that series with a longer follow-up now need to be tested to clarify the association between CD44 splice variant expression and distant metastasis or long-term prognosis.

Semiquantitative detection of abnormal cd44 transcripts in colon carcinomas by reverse transcription-polymerase chain reaction enzyme-linked immunosorbant assay (rt-PCR elisa)

Background: Abnormal expression of CD44 variant RNA has been detected in a variety of human tumors and has been shown to be a potential diagnostic marker. To date, such analysis requires time-consuming gel electrophoresis, blotting, and autoradiographic procedures, and this approach may not be suitable for routine laboratory examinations. We have developed a rapid and semiquantitative reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR ELISA) method and used it to analyze CD44 expression in colon carcinoma tissues and exfoliated cancer cells in colon luminal washings. Methods and Results: RNA was extracted from sample cells and tissues and converted to cDNA. PCR amplification products, labeled by incorporation of digoxigenin-11-dUTP, were hybridized with biotinylated probes complementary to CD44 exon 12 or to exons in the standard portion (CD44s) of the gene. Hybridized DNA complexes were immobilized on streptavidin-coated microtiter plates, and the bound PCR products were detected with a peroxidase-conjugated antibody to digoxigenin. CD44-derived PCR products were quantified by absorbance of a chromogenic reaction. Elevated expression of CD44 variant exon 12 was detected initially by Southern blot analysis in all of the 9 colon carcinoma tissues, while weak expression was observed in only 3 of 9 normal mucosas. This tumor-related differential expression was confirmed by the newly developed PCR-ELISA method. Elevated expression of CD44 exon 12 was also detected in exfoliated colonic epithelial cells from 10 of 13 carcinoma cases but not in exfoliated cells from 4 patients with inflammatory bowel disease. Conclusions: Raised expression of CD44 variant exon transcripts can be detected reliably in colonic tumor tissue and in exfoliated colonic cancer cells by a semiquantitative RT-PCR ELISA method. This was shown to be as sensitive as conventional RT-PCR using chemiluminescent detection. Therefore, CD44-based RT-PCR ELISA could facilitate detection of neoplasia in clinical specimens including colon washings and naturally micturated urine. (Mol Diagn 1996 Sep;1(3):167-173)

Accumulation of immature intron-containing cd44 gene transcripts in breast cancer tissues

Background: Disorderly expression of the CD44 gene is a characteristic feature of many common types of human malignancy. The explanation for the diversity, abundancy, and abnormally large size of the resulting transcripts is under investigation. Methods and Results: This study used reverse transcription-polymerase chain reaction (RT-PCR)/Southern blot hybridization to examine the expression of the CD44 gene in fresh tissue samples from 21 breast cancers and 11 matched non-neoplastic breast tissue specimens from the same patients. Using probes for several exons and for a noncoding region (intron 9) of the gene, it was found that the previously described elevated levels of CD44 transcripts in malignant tumors of this organ include many unusual, alternatively spliced isotypes as well as immature forms that retain this intron and probably several others. All of the exons that were tested were involved in the disorderly overexpression of this gene observed in all of the cancer tissues, and we were able to detect retention of the intron 9 sequence in 14 (67%) of the 21 tumor samples. In contrast, it was possible to detect signals in only 3 (27%) of the 11 samples from nonmalignant breast tissue with this probe, and these were extremely faint. Conclusions: These findings imply that there is a profound disorder in the regulation of production, splicing, and processing of CD44 pre-mRNA in breast cancer tissues comparable to that described in tumors of many other organs. The clinical implication of this information is that analysis of tissue samples containing borderline or suspected premalignant lesions for the presence of these molecular abnormalities, which appear to be characteristic of neoplasia, may in due course help assessment of individual patient prognosis and optimization of treatment. (Mol Diagn 1996 Sep;1(3):175-181)

Evaluation of CD44 transcription variants in human digestive tract carcinomas and normal tissues.

We generated DNA probes for CD44 variable region exons 11 (v6) to 14 (v9) and also for intronic sequences and examined the expression of aberrant CD44 transcripts in digestive tract carcinomas, colorectal adenomas and intestinal metaplasia of the stomach. The study used the reverse transcription-polymerase chain reaction/Southern blot technique. Among the probes generated, the CD44 intron 9 probe was the best for distinguishing cancer tissue from normal tissue in adenocarcinomas of the colorectum or stomach. All the colorectal adenocarcinomas revealed overexpression of CD44 variants containing the intron 9 sequence compared with the corresponding normal colorectal mucosa. The overexpression of such abnormal CD44 variants was observed from an early stage in colorectal cancer and did not correlate with nodal or distant metastatic status. In intestinal metaplasia of the stomach, aberrant CD44 transcripts with characteristic 2 or 3 peaks containing intron 9 were observed. On the other hand, in oral and esophageal squamous cell carcinomas and corresponding normal squamous epithelia, overexpression of CD44 variants with variable exons and retained intron 9 sequence was frequently observed in both cancer tissue and corresponding normal mucosa. Examination of the expression of CD44 variants in a screening panel of normal tissues from the whole body revealed that overexpression of transcripts containing exon 11 and 14 as well as of the intron 9 sequence was constantly observed in tissues with squamous epithelia like skin, oral mucosa, esophagus and uterine cervix. Expression of these variants was also found in urinary bladder, respiratory tract, pancreas and salivary glands. Our results overall indicate that detecting the overexpression of abnormal CD44 transcripts, especially ones containing the intron 9 sequence, could be a powerful indicator for the presence of adenocarcinomas in the digestive tract. However, it is not applicable for the diagnosis of malignancies originating from squamous epithelia.

Gene therapeutic approaches to primary and metastatic brain tumors: II. ribozyme-mediated suppression of CD44 expression.

Glioblastomas are highly invasive intracerebral tumors that are known to express the CD44 cell adhesion molecule. Human glioma cell adhesion and invasion in vitro may in part be mediated by the interaction of CD44 with extracellular matrix proteins. To suppress the growth and invasive effects of CD44 expression on primary brain tumors we have designed two hammerhead ribozymes as potential gene therapeutic agents. Both ribozymes designed to target exon 2 of CD44 exhibited in vitro cleavage of in vitro transcribed CD44s and CD44R1 RNAs. The anti-CD44 effect of these ribozymes results from directed RNA cleavage, requiring both a target sequence and an appropriate catalytic center. Further, following transient transfection of one of these ribozymes into the SNB-19 glioma cell line, significant in vivo cleavage activity against cellular CD44 transcripts was demonstrated by flow cytometrical analysis. These preliminary results suggest that CD44-directed hammerhead ribozymes may be useful as gene therapeutic agents.

Patterns of splice variant CD44 expression by normal human urothelium in situ and in vitro and by bladder‐carcinoma cell lines

CD44 core and splice variant exon expression was investigated in the stratified transitional epithelium of the urinary tract and in normal and malignant bladder epithelial cell lines in vitro. Antibodies against core CD44 epitopes and splice exon variants v3, v4/5, v5 and v6 showed an intense reaction in the basal and lower intermediate urothelial cell layers, which was consistently lost from the upper intermediate and superficial cell layers. Seven independent cell lines established from normal human urothelium expressed complex multiple-spliced CD44 mRNA transcripts when tested by RT-PCR and were positive with antibodies against CD44 core epitopes and splice variants v3, v4/5, v5 and v6. Of the 13 bladder-carcinoma cell lines, all were positive for CD44 core. The more differentiated cell lines had retained some splice-variant antibody reactivity and showed multiple but less complex CD44 mRNA transcript patterns, compared with normal cells. Anaplastic cell lines did not react with variant antibodies and did not contain multiple-spliced CD44 transcripts. These data suggest that loss of alternatively-spliced CD44 may reflect a selection pressure in the evolution of anaplastic bladder cancers.

Quantitative and qualitative changes in CD44 and MEL-14 expression by T cells in C57BL/6 mice during aging

Aging is associated with a decrease in the functional activity of T cells. We have explored age-related alterations in CD44 and MEL-14 expression by spleen cells bearing the Thy1.2, CD4 or CD8 antigens in C57BL/6 mice at 2, 8, 15 and 23 months of age. The membrane expression of CD44 and MEL-14 molecules can be used to distinguish naive (CD44low, MEL-14high) from preactivated/memory (CD44high, MEL-14low) T cells. Our results show that the proportion of CD4+ splenic cells begins to decrease at an intermediate age (8-month-old mice), whereas the proportion of CD8+ cells remains unaltered. The proportion of CD4+ and CD8+ splenic cells with the CD44high memory phenotype was increased at an early stage of aging (in 8-month-old mice) without a concomitant change in MEL-14 expression. In older mice, MEL-14 expression decreased on CD4+ but not on CD8+ subsets. Recent studies have reported that following activation, the expression of CD44 molecules containing additional, so-called variable exons can be detected. By PCR, we observed an increase in CD44 transcripts containing the v6 or v7 variable exons in murine lymph nodes at the age of 15 months. Our results suggest that v6- or v7-containing variants of CD44 may be involved in the development of memory cells. Taken together, these results suggest that the trafficking of memory T cells in aging may be altered by quantitative and/or qualitative differences in the expression of molecules involved in lymphocyte recirculation.

Expression of variant spliced CD44 transcripts in normal dysplastic and neoplastic cervical epithelium

Expression of variant spliced CD44 transcripts in normal dysplastic and neoplastic cervical epithelium

CD44 variant expression in human lung cancer and normal bronchial epithelium

P 12 CD44 VARIANT EXPRESSION IN HUMAN LUNG CANCER AND NORMAL BRONCHIAL EPITHELIUM M. Givehchian, S. M. Woerner, M. Z011er, P. Drings, H. Becket, K. Kayser, M. von Knebel Doeberitz. CD44 variants have been reported to confer metastatic potential in human and rat cancers. We analyzed the expression of CD44 splice variants in human lung cancer and normal bronchial epithelium. Immunohistochemical analysis shows that normal bronchial epithelial cells express a complex pattern of CD44 splice variants including variant exon 5, variant exon 6 and the standard form. Squamous cell carcinoma retains this expression pattern of CD44 splice variants. No correlation of alternativ CD44 splice pattern and lymph node or organ metastasis was observed. Interestingly highly metastatic small cell lung cancers and adenocarcinomas did not stain with the antibodies. Exon specific PCR amplification of CD44 transcripts suggests that the normal bronchial epithelium and squamous cell carcinomas express 5 major types of CD44 mRNA. The first type contains only variant exon 2. The second typ contains variant exons 3, 4 and 5. The third type contains just variant exons 4 and 5. The fourth type contains the variant exons 6 and 7. The fifth type contains variant exons 8, 9 and 10. In classic small cell lung cancer cells we did not detect expression of CD44 variant exon transcripts, whereas variant SCLC cells express the standard form of CD44. These data do not support the hypothesis that expression of specific variant CD44 molecules contributes to metastatic spread of lung cancer. They suggest that CD44 variants are expressed in a strictly differentiation dependent pattern within the bronchial epithelium.

Identification of CD44 splice variant in Korean colorectal cancers and cell lines.

CD44 is a glycoprotein expressed in a wide variety of cell types. Recently expression of some alternatively-spliced variants of CD44 transcripts (CD44v) has been suggested to play a potential role in tumor metastasis and the detection of CD44v containing exon 6 to 11 may be helpful for the diagnosis of cancers. Expressions of CD44v containing exon 6 to 11 were investigated in 20 human colorectal cancer samples, peripheral blood leukocytes isolated from colorectal cancer patients, and 4 colorectal cancer cell lines using reverse transcription-polymerase chain reaction and Southern blot analysis. The standard form of CD44 transcripts was expressed in all samples tested. CD44v containing exon 6 to 11 was expressed in 18 cases of colorectal cancers (sensitivity = 90%), 3 out of 4 cell lines, and one normal tissue (specificity = 95%). These results suggest that the expression of CD44v containing exon 6 to 11 can be regarded as tumor specific and that this marker may be helpful for the early diagnosis of colon cancers, if specimens from the early stage are available.

Alternative Splicing of CD44 pre-mRNA in Human Colorectal Tumors

Expression of the CD44 molecule has been linked to tumor growth and metastases in both human and rodent cancers. Alternatively spliced variants expressed in rat and mouse tumors have been shown to confer metastatic potential to non-metastatic carcinoma cell lines, and human homologues of rat variant mRNA sequences are expressed in human tumors. In the present study matched sets of RNA from adenocarcinomas of the colon and distant normal mucosa were assayed for CD44 expression by quantitative RT-PCR. Retrospective analysis revealed that colonic tumor cells had both quantitative and qualitative differences in CD44 expression when compared to normal mucosa. These were: 1) an increase in levels of CD44 transcripts, 2) an increase in levels of alternatively spliced transcripts, 3) the presence of larger alternatively spliced transcripts with inserts >400 bases and 4) the primary alternatively spliced CD44 isoform in colonic adenocarcinomas in all cases is CD44R. Interestingly, two patterns of CD44 isoform expression termed "variant dominant" or "balanced" patterns of expression, based on the ratio of variant to standard CD44 transcripts (R+V′s/H), could be differentiated. An unfavorable prognosis was suggested for tumors expressing increased levels of CD44 variant exons previously associated with tumor metastasis. Specifically, patients with tumors expressing the "variant dominant" pattern of expression irregardless of Dukes classification and Dukes C and D staged tumors of both patterns exhibited a poorer prognosis.

Alternatively Spliced CD44 Transcripts in Diffuse Large-Cell Lymphomas: Characterization and Comparison With Normal Activated B Cells and Epithelial Malignancies

AbstractCD44 exists in a variety of alternatively spliced isoforms that include variable numbers of additional exons from the membrane proximal domain (exons 6-14). Lymphocytes express high levels of a “hematopoietic” isoform (CD44H) with no additional variable exons. CD44H binds lymphocytes to postcapillary venules and promotes lymphocyte extravasation into nodal areas. Epithelial carcinomas also express CD44 variants, including exon 10-containing isoforms associated with metastasis in a rat model. An exon 10-containing CD44 isoform is also transiently expressed by normal activated lymphocytes, suggesting that the protein may function in the trafficking of both normal lymphocytes and metastatic tumors. To identify the specific CD44 transcripts in tumors from patients with primary nodal, extranodal, and disseminated large-cell lymphomas (LCLs) and compare them with CD44 mRNAs in normal Ig-activated splenic B cells and epithelial cells, we used a semiquantitative RNA-based polymerase chain reaction. Specific CD44 variants were identified by size, hybridization with exon-specific probes, and sequence analysis. In comparison to primary nodal LCLs, extranodal and disseminated LCLs had increased levels of CD44H and additional isoforms, including a directly spliced (exon 5 → exon 10 → exon 15) exon 10-containing CD44 variant. The CD44 transcripts in extranodal and advanced-stage LCLs were similar to those in normal lg-activated splenic B cells, whereas epithelial malignancies contained decreased CD44H and increased amounts of larger CD44 variants with additional exons from the membrane proximal domain. The regulated expression of specific CD44 variants is likely to influence the trafficking of lymphoid and epithelial malignancies.