Expression Of CD44 Splice Variants Research Articles

3332 Overexpression of CD44 is involved in the development of the early endometriotic lesion in a xenograft model

OBJECTIVES/SPECIFIC AIMS: Previously, we showed decreased development of endometriotic lesions in CD44 knockout mice compared to control.(1) CD44 has 10 different variants and a standard form. Menstrual endometrial cells (MECs) from women with endometriosis have increased adhesion and also express higher levels of CD44 variant 6 (v6) than v3, compared to MECs from women without endometriosis. (2) Here, we assessed the effects of CD44 standard (CD44s), CD44v3 and CD44v6 overexpression (OE) on immortalized human endometrial epithelial (iEECs) and stroma cells (hESCs) in vivo attachment in a nude mouse xenograft model. 1. Knudtson JF, Tekmal RR, Santos MT, et al. Impaired Development of Early Endometriotic Lesions in CD44 Knockout Mice. Reproductive sciences (Thousand Oaks, Calif.). 2016;23(1):87-91. 2. Griffith JS, Liu YG, Tekmal RR, Binkley PA, Holden AE, Schenken RS. Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants. Fertility and sterility. 2010;93(6):1745-1749. METHODS/STUDY POPULATION: Overexpression of CD44s, CD44v3 and CD44v6 was carried out using lipofectamine and their expression verified with qRT-PCR in iEEC and hESCs. Nude mice, 8-10 week old, were injected with estrogen 1 week prior to injection of iEECs and hESCs (n=7 per group). The cells were counted after transfection and at least 300,000 iEECs and 300,000 hESCs were injected per mouse. The transfected cells were tagged with cell tracker red (iEECs) and green (hESCs). Forty-eight hours after injection into the xenograft, the mice were sacrificed. The cells were counted using fluorescent stereo microscopy (FSM). Percent attachment was calculated based on the number of cells visualized by FSM divided by the number of transfected cells injected. Unpaired student t-test was performed to analyze differences in the percent attachment of the cells. RESULTS/ANTICIPATED RESULTS: The majority of cells were attached to the peritoneum. There was increased attachment of hESCs with OE of CD44v6 compared to control (p=0.03). CD44v6 OE did not change attachment of iEECs. There was no difference in attachment in iEECs or hESCs with OE of CD44s or CD44v3. DISCUSSION/SIGNIFICANCE OF IMPACT: Overexpression of CD44v6 increases attachment of ESCs to PMCs in an in vivo xenograft model. Menstrual endometrial cell type and CD44 variants play a complex role in the development of the early endometriotic lesion.

Malignant Reprogramming of Progenitors into Leukemia Stem Cells Is Enhanced By Upregulation of CD44 Transcript Variant 3 in Malignant Microenvironments

IntroductionMalignant reprogramming, first described in chronic myeloid leukemia (CML), occurs upon activation of the Wnt/b-catenin pathway in granulocyte-macrophage progenitors (GMPs) that gain the capacity to self-renew and contribute to the emergence of BCR-ABL1 tyrosine kinase inhibitor (TKI) resistant blast crisis CML. Deregulation of the Wnt/b-catenin target gene, CD44, plays a vital role in leukemia stem cell (LSC) maintenance in the malignant microenvironment in mouse models of CML. However, extensive alternative mRNA splicing in humans results in expression of multiple CD44 isoforms, some of which have been implicated in cancer invasion and metastasis. In this study we investigated the role of CD44 splice variant expression on human blast crisis LSC maintenance in the malignant niche.Methods and ResultsCD44 Isoform Expression AnalysisTo investigate the splice isoform expression pattern of CD44, whole transcriptome RNA sequencing (RNA Seq; Illumina HiSeq 2000) was performed on FACS sorted chronic phase (CP; n=8) and blast crisis (BC; n=8) CML progenitors (CD34+CD38+Lin-) as well as their normal counterparts from cord blood (CB) (n=7) and adult peripheral blood (NPB; n=4). While whole gene expression analysis revealed upregulation of CD44 in blast crisis compared with chronic phase and normal progenitors, a plethora of CD44 transcript variants were also detected including variants 3, 4 (CD44s), 5, 6, 7, 8. Notably, RNA Seq isoform analysis detected a higher expression of CD44 transcript variant 3 in BC compared to CP and CB and NPB. Moreover, CD44 transcript variant 3 gene expression was highly expressed in undifferentied human embryonic stem cells (hESCs) while differentiated hESCs (embryoid bodies) had low expression, suggesting CD44 transcript variant 3 to be important for pluripotency.Lentiviral CD44 Variant 3 OverexpressionTo directly determine the impact of CD44 variant 3 expression on malignant reprogramming of CP progenitors into self-renewing LSC, we developed a lentiviral human CD44 variant 3 overexpression vector and transduced CP CML progenitors. Transduced CP progenitors harbored increased expression of migration specific markers, such as osteopontin and ICAM1, as well as an upregulation of the pro-survival long isoforms of BCL2 family members BCLX and MCL1, thereby enhancing survival and replating in hematopoietic progenitor assays. Moreover, hESCs transduced with CD44 transcript variant 3 showed upregulation of pro-survival BCL2 isoforms, enhanced proliferation and as well as maintenance of an undifferentiated state, suggesting that CD44 transcript variant 3 promotes pluripotency.Targeted Inhibition of CD44 variant 3 Expressing LSCHumanized RAG2-/-gc-/-mice engrafted with CD34+ BC CML patient samples showed a significant reduction of human progenitor cells post treatment with a clinical grade CD44 mAb, both alone and in combination with Dasatinib in all hematopoietic niches. Bone marrow and spleen samples from primary transplanted mice show a reduced gene expression level of CD44 and CD44 transcript variant 3 upon combination treatment of CD44 and Dasatinib. Most importantly, serial transplantation of progenitors treated with the CD44 mAb as well as in combination with Dasatinib revealed a significant reduction in LSC self-renewal capacity commensurate with a reduction in CD44 variant 3 expression.ConclusionsUpregulation of an embryonic splice variant of CD44, variant 3, expands pluripotent stem cell populations and promotes malignant reprogramming of CML progenitors into self-renewing LSC. Treatment with a humanized CD44 specific mAb sensitizes CML LSC residing in malignant niches to Dasatinib. From these results CD44 mAb appears to be an excellent antibody for future combination clinical studies aimed at eradicating therapy resistant blast crisis LSC in CML. In addition, these observations strongly suggest that CD44 transcript variant 3 upregulation serves as a biomarker of progression from CP to BC as well as the generation of TKI resistant LSCs, with the potential of being a more specific target for future combination therapies. DisclosuresNo relevant conflicts of interest to declare.

Abstract 211: Sulfasalazine (SSZ) works cancer stem-like cells (CSCs) via inhibiting xCT signal pathway: Phase 1 study in patients with gastric cancer (EPOC 1205)

Abstract Back ground: CD44 is an adhesion molecule expressed in cancer stem-like cells (CSCs). Our group recently reported that CD44 splice variant (CD44v) is expressed in CSCs and interacts with xCT, a glutamate-cystine transporter, keeping high levels of the intracellular reduced glutathione (GSH). Thus, CSCs with a high expression of CD44v have an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS), resulting in resistance to various therapeutic stresses (Cancer Cell 2011; Cancer Res 2013). Sulfasalazine (SSZ) as an xCT inhibitor suppressed CD44v-dependent tumor growth and increased sensitivity to cytotoxic drugs in vivo study. Methods: A phase 1 dose escalation study in patients with advanced gastric cancer who had received one or more standard chemotherapies was conducted to determine the optimal dose, change in CD44v expression and intra-tumor level of GSH pre- and post SSZ exposure and pharmacokinetics. SSZ was given fourth-daily oral administration with 2 weeks as one cycle, which were continued until progression of disease, unacceptable toxicity, or other discontinuation criteria were met. A 3+3 escalation was used to evaluate a MTD. Tumor tissues were obtained pre- and post SSZ administration to evaluate expression of CD44v and intra-tumor level of GSH by immunohistochemistry and boron doped diamond microelectrode (Sci Rep 2012), respectively. Results: Eleven patients were dosed from 8 g to 12 g/day; median age: 71 years (61-78); median number of prior chemotherapies: 3 (1-4). There was two DLT of grade 3 anorexia and nausea among patients who were treated with 12 g/day. One additional patients required frequent dose interruption with grade 2 anorexia and nausea. Therefore 12g/day was judged as MTD. No DLT was observed among patients with 8g/day. Among the 5 patients with high CD44v expression, 4 patients achieved reduced expression of CD44v after the administration of SSZ for 2 weeks. GSH was also apparently decreased in 2 of 5 patients. Further exploratory biomarker analyses are still ongoing. The individual variability of SSZ exposure was explainable in terms of the genotypes of ABCG2 and NAT2 which influence SSZ pharmacokinetics. Conclusions: Optimal dose of SSZ was considered as 8g/day. Down regulation of CD44v expression and decreased level of GSH is confirmed as a pharmacodynamic marker of drug-on-target effect and mode of action of SSZ for CSCs, which warrants further investigation for combination with chemotherapy or other targeting agents. Clinical trial information: UMIN000010254 Citation Format: Kohei Shitara, Shunji Takahashi, Takako Nakajima, Hironaka Shuichi, Osamu Nagano, Chiyo Imamura, Taisei Mushiroda, Yasuaki Einaga, Miki Fukunani, Akihiro Sato, Atsushi Ohtsu, Hideyuki Saya, Toshihiko Doi. Sulfasalazine (SSZ) works cancer stem-like cells (CSCs) via inhibiting xCT signal pathway: Phase 1 study in patients with gastric cancer (EPOC 1205). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 211. doi:10.1158/1538-7445.AM2014-211

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy that may involve the oral cavity, pharynx, larynx, and paranasal sinuses. The mechanisms of tumor progression underlying the clinical behavior of HNSCC remain unclear. CD44 comprises a family of transmembrane receptors that can give rise to multiple CD44 variant isoforms. Hyaluronan (HA), a major extracellular matrix component is the primary ligand for CD44 receptors. HA and CD44 signaling play an important role in HNSCC progression. Several CD44 variant isoforms (including v3-, v6-, and v10-containing isoforms) are associated with advanced disease, possibly through unique growth factor interactions with binding domains in the inserted variant regions of the cytoplasmic domain of CD44. In HNSCC, HA mediates the formation of a complex including CD44 and the epidermal growth factor receptor (EGFR) which is overexpressed in a large proportion of HNSCCs. Downstream effectors under EGFR regulation are activated, promoting promote cell growth and tumor survival. The leukemia-associated Rho-guanine nucleotide exchange factor (LARG) also associates with CD44 and EGFR to promote several Ras and RhoA pathway effectors, leading to cell migration, growth, and tumor survival. The secretion of matrix metalloproteinases, necessary for tumor cell invasion, is also regulated by these HA/CD44-mediated pathways. Finally, EGFR-mediated pathways play major roles in the HA/CD44 promotion of chemoresistance in HNSCC. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment of HNSCC.

Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants

Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants

Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants

We previously demonstrated that adherence of endometrial epithelial (EECs) and stromal cells (ESCs) to peritoneal mesothelial cells (PMCs) is partly regulated by ESC/EEC CD44 interactions with PMC associated hyaluronan. CD44, a transmembrane glycoprotein and major ligand for hyaluronan, has numerous splice variants which may impact hyaluronan binding. Here, we assessed whether ESCs and EECs from women with endometriosis demonstrate increased adherence to PMCs and examined CD44 splice variants' potential role in this process. In vitro study. Academic medical center. Fertility patients with and without endometriosis. Menstrual endometrium was collected from women with and without endometriosis confirmed surgically. The adherence of ESC/EECs to PMCs was measured. The ESC/EEC CD44 splice variants were assessed using dot-blot analysis. The ESCs and EECs from women with endometriosis demonstrated increased adherence to PMCs. The predominant CD44 splice variants expressed by ESCs and EECs from women with and without endometriosis were v3, v6, v7, v8, v9, and v10. The ESCs and EECs from women with endometriosis were more likely to express v6, v7, v8, and v9. Increased eutopic endometrial-PMC adherence and CD44 splice variant expression may contribute to the histogenesis of endometriotic lesions. Elucidation of factors controlling this expression may lead to novel endometriosis therapies.

Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants

Using an in vitro model for the early endometriotic lesion, we previously demonstrated that adherence of endometrial epithelial (EECs) and stromal (ESCs) cells to peritoneal mesothelial cells (PMCs) is regulated in part by ESC/EEC surface CD44 interactions with hyaluronan produced by PMCs. CD44, a transmembrane glycoprotein that is the major ligand for hyaluronan, has numerous splice variants and some are associated with an increased ability to bind hyaluronan. Here, we assessed whether ESCs/EECs from women with endometriosis demonstrate increased adherence to PMCs and examined the potential role of CD44 splice variants in this process. In vitro study. Endometrium was collected at the time of menses from women with (N = 21) and without (N = 8) endometriosis (controls) as confirmed surgically. EEC/ESC adherence to the PMC line LP9 was compared using an established in vitro assay. CD44 splice variants were assessed in ESCs/EECs using exon specific hybridization with dot blot analysis. ESCs from women with endometriosis demonstrated increased adherence to PMCs when compared to ESCs from controls (43% vs. 32%, P<0.002). EECs from women with endometriosis also demonstrated an increased adherence to PMCs that approached statistical significance when compared to EECs from controls (22% vs. 15%, P=0.07). The predominant CD44 splice variants expressed by ESCs and EECs from women with and without endometriosis were v3, v6, v7, v8, v9, and v10. ESCs from women with endometriosis were more likely to express v6, v7, v8 or v9 than ESCs from controls (72% vs. 45%, P<0.04). Increased expression of these variants by EECs from women with endometriosis approached statistical significance when compared to controls (94% vs. 75%, P=0.06). The present study is the first to demonstrate that menstrual ESCs from women with endometriosis have increased adherence to PMCs when compared to ESCs from controls. The increased expression rates of v6, v7, v8, or v9 by ESCs from women with endometriosis suggest that qualitative differences in ESC CD44 isoform expression may contribute to the pathogenesis of endometriosis. Similar findings of increased adherence and CD44 isoform expression by EECs from women with endometriosis approached statistical significance. Studies are in progress to further clarify the role of variable CD44 isoform expression in the genesis of the early endometriotic lesion and its potential for future targeted therapies.

Difference in expression of CD44 splice variants between proximal and distal adenocarcinoma of the large bowel.

To help determine whether adenocarcinomas of the proximal and distal large bowel reflect distinct entities, the expression of two splice variants of the metastasis-associated cell adhesion molecule CD44, carrying exons v5 and v6 respectively, was investigated retrospectively in fresh frozen samples of 23 proximal and 41 distal carcinomas by immunohistochemical staining with specific anti-CD44v5 monoclonal antibody VFF8 and anti-CD44v6 monoclonal antibodies VFF4 and VFF7. Tumours were staged as: Dukes A, 0; Dukes B, 27; Dukes C, 31; and Dukes 'D', six. Compared with distal tumours, proximal lesions expressed significantly more CD44v5 (96 versus 87 per cent, P = 0.02) and CD44v6 (83 versus 61 per cent, P = 0.01). CD44v5 and CD44v6 are considered markers for tumour progression and aggressive behaviour. Their high expression in proximal carcinomas seems to contrast with the general belief that these tumours show less aggressive behaviour than left-sided lesions. Further study of the biological significance of the expression of CD44 splice variants is therefore needed.

Regulation of proximal tubular epithelial cell CD44-mediated binding and internalisation of hyaluronan

Background: Increased expression of the connective tissue polysaccharide hyaluronan (HA) in the renal corticointerstitium is associated with progressive renal fibrosis. Numerous studies have demonstrated involvement proximal tubular epithelial cells in the fibrotic process and in the current study we have characterised their expression of the HA receptor, CD44, and examined changes in CD44 expression and function in response to either IL-1β or glucose. Methods: Characterisation of CD44 splice variant expression was carried out in primary cultures of human proximal tubular cells (PTC) and HK2 cells. Binding and internalisation HA was examined by addition of exogenous of fluorescein-HA (fl-HA), and expression of CD44 examined by immunoblot analysis and flow cytometry. Alteration in “functional” CD44 was determined by immunoprecipitation of CD44 following stimulation in the presence of fl-HA. Results: PTC, both primary culture and the PTC cell line, HK2, express at least 5 CD44 splice variants, the expression of which are not altered by addition of either IL-1β or 25 mM d-glucose. Addition of either stimulus increased cell surface binding and internalisation of fl-HA and increased expression of functionally active CD44. Increased binding and internalisation of fl-HA, was blocked by anti-CD44 antibody, and by the inhibition of O-glycosylation. Conclusions: The data demonstrate that stimuli inducing PTC HA synthesis also regulate PTC–HA interactions. Furthermore increased HA binding and internalisation is the result of post-translational modification of CD44 by O-glycosylation, rather than by alteration in expression of CD44 at the cell surface, or by alternate use of CD44 splice variants.

Influence of epitopes CD44v3 and CD44v6 in the invasive behavior of fibroblast-like synoviocytes derived from rheumatoid arthritic joints.

To investigate the functional implications of CD44 splice variant expression in fibroblast-like synoviocytes (FLS) obtained from patients with rheumatoid arthritis (RA). FLS were isolated from synovial tissue obtained from both diseased and nondiseased joints. The expression of splice variants containing exons v3 and v6 was analyzed using immunocytochemistry with exon-specific antibodies and reverse transcription-polymerase chain reaction followed by Southern blotting. The invasive capacity of the cells was studied in a transwell invasion assay. FLS obtained from RA joints expressed various CD44 splicing combinations containing the variant exons v3 and/or v6. These cells were highly invasive, whereas cells from normal tissues, which lacked expression of CD44 splice variants, were not. Variant exons CD44v3 and CD44v6 were instrumental in matrix invasion in vitro, with cells enriched for CD44v3 and v6 exhibiting greater invasion and antibodies that specifically recognize CD44v3 and v6 abrogating this capacity to invade. Invasive cells showed a reduced expression of CD44v7/8, and antibodies against this epitope had no significant effect on cellular infiltration of the matrix. The antibodies had no effect on cell migration into the porous section of the transwell. FLS obtained from patients with RA express CD44 splice variants and are highly invasive, whereas cells obtained from healthy tissue do not express these variants and are not invasive. Expression of the epitopes CD44v3 and CD44v6 is instrumental in the invasive capacity but not in cell migration. This finding highlights a functional implication for the expression of CD44 splice variants at the level of matrix degradation.

Expression of CD44 splice variants in spontaneous murine tumors.

CD44 is a widely distributed set of cell surface glycoproteins expressed in several types of cells and tissues, implicated in cell-cell and cell-substrate interactions. This molecule plays a major role in cell differentiation, development and activation and has also been described as a potential marker of malignancy and metastasis. In the present study we investigated by RT-PCR followed by exon specific amplification the expression of CD44 splice variants in four different murine tumors as well as in the invaded organs in order to correlate the expression of CD44 variants with potential tumor invasiveness and their implications for growth. Our data showed deregulation in the expression of CD44 isoforms but no discernible correlation in isoform expression pattern. However, in all tumors studied isoforms presented by the primary tumor were detected in the invaded organs before metastasis could be demonstrated by histopathological analysis.

Prognostic Value of CD44 Expression in Invasive Squamous Cell Carcinoma of the Vulva

Objective. CD44 is a cell adhesion molecule that binds extracellular matrix. CD44 isoforms arising from alternative mRNA splicing are implicated in tumor metastases. The aim of our study is to investigate the expression of CD44 splice variants and its correlation to lymph node metastases and disease-free survival in squamous cell carcinoma (SCC) of the vulva.Methods. Thirty-five cases of SCC of the vulva were evaluated for CD44 splice variants -3v, -4v, -5v, and -7v expression by immunocytochemistry. When available one nonmetastatic lymph node (LN) was also studied. In cases with LN metastases, the metastatic LN as well as a nonmetastatic LN from the same patient were evaluated.Results. All CD44 variants studied were expressed in all epithelium: normal, dysplastic, and SCC. CD44 variants showed decreased immunostaining in the tumor cells when compared to normal epithelium. Furthermore, intensity of expression of the CD44 isoforms changed within the tissue containing invasive cancer. Interestingly, CD44-4v expression was downregulated in the most differentiated cells within the carcinoma, mainly in patients who had disease recurrence or died of disease (P = 0.004). Confirming prior publications, CD44-5v and -7v expression did not correlate with survival. One hundred percent of metastatic tumors to LNs were immunoreactive with CD44-3v and only 1/30 normal LN had CD44-3v expression. Eighty percent of metastatic tumors to LNs were immunoreactive for CD44-4v. However, 3 LNs without tumor were also immunoreactive with CD44-4v.Conclusion. CD44-4v is a potential molecular marker of disease recurrence in vulvar carcinoma. A larger multiinstitutional study is needed to evaluate the specificity of CD44-3v expression in LN metastasis. If a larger scale study confirms our findings, a CD44-3v antibody could be used for radioimmunoimaging of occult lymph node metastases in patients with vulvar cancer.

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

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

Highly specific and sensitive detection of malignancy in urine samples from patients with urothelial cancer by CD44v8-10/CD44v10 competitive RT-PCR.

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.

Expression of CD44 splice variant v10 in Hodgkin's disease is associated with aggressive behaviour and high risk of relapse

Expression of CD44 isoforms has been shown to correlate with the progression and prognosis of some malignant tumours. The aim of this study was to investigate the expression of CD44 standard (CD44s) and CD44 splice variants (CD44v) v5, v6, and v10 in lymph node specimens from patients with nodular sclerosing Hodgkin's disease (NSHD), with or without initial bone marrow involvement and with or without relapse. Specimens were studied by immunohistochemistry to determine CD44s and CD44v in Hodgkin- and Reed–Sternberg (HRS) cells. For validation of the immunohistochemical detection of CD44v10 in paraffin-embedded samples, selected cases were analysed in parallel immunohistochemically using fresh frozen material and by reverse transcription-polymerase chain reaction (RT-PCR). There was high expression of CD44 isoforms containing the variant exon v10 selectively in HRS cells of patients with relapse within 2–3 years or with initial bone marrow involvement. In patients without relapse, however, no or only very few HRS cells were positive. These differences were statistically highly significant (p≤ 0001), whereas evaluation of CD44s, CD44v5, and v6 expression revealed no marked differences. It is concluded that evaluation of CD44v10 expression could serve as a new prognostic marker in NSHD. These results are considered to be of sufficient importance to initiate a large multi-institutional study for confirmation; furthermore, they might suggest causal involvement of CD44v10 in the progression of NSHD. Copyright © 1998 John Wiley & Sons, Ltd.

Expression of CD44 splice variant v10 in Hodgkin's disease is associated with aggressive behaviour and high risk of relapse.

Expression of CD44 isoforms has been shown to correlate with the progression and prognosis of some malignant tumours. The aim of this study was to investigate the expression of CD44 standard (CD44s) and CD44 splice variants (CD44v) v5, v6, and v10 in lymph node specimens from patients with nodular sclerosing Hodgkin's disease (NSHD), with or without initial bone marrow involvement and with or without relapse. Specimens were studied by immunohistochemistry to determine CD44s and CD44v in Hodgkin- and Reed-Sternberg (HRS) cells. For validation of the immunohistochemical of detection of CD44v10 in paraffin-embedded samples, selected cases were analysed in parallel immunohistochemically using fresh frozen material and by reverse transcription-polymerase chain reaction (RT-PCR). There was high expression of CD44 isoforms containing the variant exon v10 selectively in HRS cells of patients with relapse within 2-3 years or with initial bone marrow involvement. In patients without relapse, however, no or only very few HRS cells were positive. These differences were statistically highly significant (p < or = 0.001), whereas evaluation of CD44s, CD44v5, and v6 expression revealed no marked differences. It is concluded that evaluation of CD44v10 expression could serve as a new prognostic marker in NSHD. These results are considered to be of sufficient importance to initiate a large multi-institutional study for confirmation; furthermore, they might suggest causal involvement of CD44v10 in the progression of NSHD.

CD44 Splice Variant Expression in Clear Cell Carcinoma of the Ovary

Objective.CD44 is a surface glycoprotein widely distributed among different tissues. Malignant tumors may show a more complex pattern of CD44 expression, indicating a loss of splice control. The aim of our study is to investigate the expression of CD44 splice variants (CD44v) and its metastatic potential in clear cell carcinoma of the ovary.Methods.Twenty-two cases of clear cell carcinoma of the ovary were evaluated for CD44 standard form (CD44s) and splice variants: -4v, -6v, and -9v expression by immunocytochemistry.Results.Twenty-one primary ovarian tumors and 23 metastatic sites were available for evaluation. Eighteen of 21 (86%) of ovarian sections studied expressed CD44s; 15/21 (71%) expressed CD44-4v; 14/21 (67%) expressed CD44-6v; and 12/21 (57%) expressed CD44-9v. Of 23 metastatic sites evaluated, 87% expressed CD44s. In contrast, only 5 (22%) metastases had CD44-4v and CD44-6v expression and 8 (35%) had CD44-9v immunoreactivity. None of 10 normal contralateral ovaries expressed CD44s or any splice variants. In 2 cases we had tumor available from the primary surgery, and subsequent recurrences. Both recurrences showed decreased expression of CD44-4v and CD44-6v.Conclusions.Clear cell carcinoma of the ovary shows an abnormal pattern of CD44s expression and mRNA splicing when compared to the contralateral normal ovary in the same patient. Metastases of clear cell carcinoma show a downregulation in expression of some splice variants. Furthermore, we have data that suggest that as the tumors recur, CD44s and its isoforms are downregulated. Our results suggest that alternative mRNA splicing of CD44 may be important in the development of metastases from clear cell carcinoma of the ovary.

CD44 splice variant expression in normal and malignant uterine cervical epithelium

CD44 expression was investigated immunohistochemically on paraffin sections obtained from 88 uterine cervical cancers and 31 normal cervices, using monoclonal antibodies against CD44 variant epitopes v4, v5, v6, v7/8, v9 and the standard form of the CD44 protein. Normal epithelium showed expression of all CD44 splice variants, at least in traces, and it was located predominantly in basal and parabasal cells. In cervical carcinomas CD44 expression was widely heterogeneous. CD44 variant v9 staining was moderate or strong in 86% of the tumors, and it was significantly correlated with CD44 v6 staining. Also a significant correlation between expression of CD44 v4 and v6 occurred. Highly significant correlations between CD44 expression of variants v4 and v6 and tumor stage as well as patients age were found. In addition, these variants were more frequently expressed in squamous cell carcinomas than in adenocarcinomas. However, in contrast to the recently reported data, we were not able to confirm the hypothesis that CD44 v6 represents a prognostic indicator in cervical cancer. In FIGO stages III and IV, patients with CD44 variant v4 positive tumors had a significantly longer disease-free and overall survival than patients with CD44 variant v4 negative tumors. In conclusion, our data indicate that CD44 v6 tissue expression cannot be considered as a prognostic factor in cervical cancer. Regarding the unexpected outcome of patients with CD44 v4 positive tumors, further investigations are needed to elucidate the exact clinical value of this variant isoform.

Expression of CD44 and isoforms v4, v5, v6, v7, v10--new prognostic parameters in ductal pancreatic carcinoma?

Prognostically relevant factors based on the histological assessment of the resected pancreas are known. However, additional parameters, such as biological staging of the intrinsic malignant potentiality of the tumor, would be useful. There has been no uniform finding of a relationship between CD44 variant expression and tumor progression. We assessed the expression pattern and prognostic impact of CD44 standard and CD44 isoforms v4, v5, v6, v7 and v10 in 40 ductal pancreatic carcinomas by immunochemistry on paraffin-embedded tumor material in patients after tumor resection. At the time of the investigation neither histomorphological parameters nor the survival time were known. There was no correlation between the histomorphological parameter and the expression of CD44 splice variants. CD44 splice variants v4-v10 were almost only expressed in tumor tissue. In ductal pancreatic carcinoma, patients with an overexpression of CD44 splice variants had a worse prognosis. However, because of the small number of cases this was statistically not significant. CD44 splice variants may have an influence on prognosis in ductal pancreatic carcinoma. However, further investigation on a larger number of patients is necessary to confirm these results.

Expression of CD44 variant proteins in adenocarcinoma of Barrett's esophagus and its relation to prognosis.

None of the commonly used staging criteria accurately determine the prognosis of a patient with adenocarcinoma of Barrett's esophagus. The authors therefore assessed the expression pattern and prognostic impact of CD44 standard and CD44 isoforms CD44v4, v5,v6,v7, and v10 in adenocarcinoma of Barrett's esophagus. Specimens from 41 patients with adenocarcinoma of Barrett's esophagus who underwent esophageal resection were embedded in paraffin and studied immunohistochemically to determine the expression of CD44 splice variants. Histomorphologic parameters and survival time were not known at the time of the investigation. Correlations between favorable clinical or histomorphologic parameters and CD44s or any of the split variants could not be established. Down-regulation of CD44s and the split variant v10 was significantly correlated with pT classification. Furthermore, down-regulation of CD44v10 and up-regulation of CD44v7 were significantly correlated with ploidy. There was a significant correlation between CD44s and split variants in tumorous and nontumorous tissue from the same patient. Down-regulation of CD44s and CD44v4 had a significant influence on prognosis in that it was associated with shortened life expectancy. Multivariate analysis revealed that the expression of CD44v4 was an independent factor in prognosis. The results obtained for this small patient sample suggest that CD44v4 is a new independent prognostic parameter for adenocarcinoma of Barrett's esophagus that can be determined preoperatively by biopsy. It may therefore be helpful in planning therapy by allowing the identification of patients who may benefit from esophageal resection as well as those who are at high risk for morbidity and mortality even when the tumor is otherwise resectable. Further studies of larger patient samples are required to validate the results of the current study.