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Overexpression of CD44 Variants 6 and 7 in Human Endometrial Cancer
GYNECOLOGIC ONCOLOGY ARTICLE NO.
71, 185–189 (1998)
GO985169
Overexpression of CD44 Variants 6 and 7 in Human Endometrial Cancer Masumi Katsura, M.D.,1 Hiroyuki Furumoto, M.D., Masato Nishimura, M.D., Masaharu Kamada, M.D., and Toshihiro Aono, M.D. Department of Obstetrics and Gynecology, University of Tokushima School of Medicine, 3-18-15 Kuramoto, Tokushima 770, Japan Received April 1, 1998
CD44 is broadly expressed on many types of epithelial cells as well as on lymphocytes [3, 4]. There are at least 17 variant isoforms (CD44V) generated by alternative splicing of at least 10 exons [5]. A variant form of CD44 has recently been found to affect metastatic potential of cancer cells. Gunthert et al. [6] reported that CD44V containing V4 –V7 were expressed only in metastatic cell lines, and not in nonmetastatic cell lines. Furthermore, overexpression of this variant isoform is sufficient for the nonmetastasizing rat pancreatic carcinoma cells to acquire metastatic potential. CD44V expression has been demonstrated in many types of human cancers. Expression of CD44V is increased in non-Hodgkin’s lymphoma [7, 8], breast cancer [9], colon cancer [10, 11], and gastric cancer [12, 13] compared with corresponding normal tissues. Furthermore, CD44V expression has been correlated with lymph node metastasis in colon cancer [10], gastric cancer [12, 13], and breast cancer [14]. On the other hand, in prostate cancer [15, 16] and squamous cell carcinoma of the head and neck [17], expression of CD44V is decreased compared with corresponding normal tissues, and reduced CD44V expression is associated with tumor metastasis. The marked differences in CD44V expression by histological type suggest that the functions of CD44V may differ by histological type. Indeed, from the clinical point of view, several papers have reported a correlation between metastatic potential or prognosis and CD44 V6, V7, and V10 [10, 18]. However, very little is known about the expression of CD44V in endometrial cancer. In this study, we examined the expression of CD44 V6, V7, and V10 in normal endometrium and endometrial cancer.
Objectives. The expression of CD44 V6, V7, and V10 in normal endometrium and endometrial cancer was compared. Methods. Using reverse transcription polymerase chain reaction (RT-PCR) blot analysis, the expression of mRNA containing CD44 V6, V7, and V10 was determined in 19 normal endometrium and 27 endometrial cancer samples. Immunohistochemical staining of CD44 V6 and V7 was performed in the same samples. Results. In RT-PCR analysis, the CD44 variant forms containing V6 and V7 exons were expressed in 96 and 93% of endometrial cancer tissues, respectively. These proportions were significantly higher than those in normal endometrium (V6, 63%; V7, 58%) (P < 0.01). CD44 V10 was expressed in 96% of endometrial cancers and 89% of normal endometrial samples. In immunohistochemical staining, CD44 V6 and V7 were detected in 48 and 61% of endometrial cancers and in 26 and 42% of normal endometrial samples, respectively. Neither of these differences was significant. No correlation was found between the expression of CD44 variants and any clinicopathological features. Conclusion. CD44 V6 and V7 were expressed in a significantly larger proportion of endometrial cancers than normal endometrial samples. However, they were also expressed in a considerable proportion of normal endometria. These findings suggest that CD44 V6 and V7 play roles in normal endometrial function and overexpression of CD44 V6 and V7 is not related to the metastatic potential of endometrial cancer. © 1998 Academic Press Key Words: CD44 variants; endometrial cancer; endometrium; reverse transcription polymerase chain reaction; immunohistochemistry.
INTRODUCTION Stage I and II endometrial cancer has been shown to have a good clinical course. However, if extrauterine metastases occur, the prognosis is very poor. Although degree of myometrial invasion and histological grade have been reported as predictors of metastasis [1], an additional predictor of metastasis would aid in the clinical management of patients with endometrial cancer. CD44 is a cell surface adhesion molecule that was originally reported as a lymphocyte homing receptor [2]. Moreover, 1
To whom reprint requests should be addressed. Fax: 81– 886 –31–2630. E-mail: [email protected]. 185
MATERIALS AND METHODS Tumor and Normal Tissue Samples Nineteen normal endometrial tissue samples (ages 27–76; 10 premenopausal, 9 postmenopausal) and twenty-seven samples of endometrial carcinoma (ages 28 –76; 1 premenopausal, 26 postmenopausal) were obtained from patients at surgery. The 27 samples comprised 21 adenocarcinomas, 4 adenoacanthomas, 1 adenosquamous carcinoma, and 1 clear cell carcinoma. 0090-8258/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Expression of CD44S and CD44V in Normal Endometrium and Endometrial Cancer
Normal endometrium Premenopausal Postmenopausal Endometrial cancer Premenopausal Postmenopausal a
CD44S (%)
V6 (%)
V7 (%)
V10 (%)
19/19 (100) 9/9 10/10 27/27 (100) 1/1 26/26
12/19 (63) 6/9 6/10 26/27 (96)a 1/1 25/26
11/19 (58) 6/9 5/10 25/27 (93)a 1/1 24/26
17/19 (89) 8/9 9/10 26/27 (96) 1/1 25/26
P , 0.01, Fisher’s exact test, compared with normal endometrium.
All samples were frozen in liquid nitrogen immediately after resection and stored at 280°C until use. Adjacent sections were fixed in paraformaldehyde and embedded in paraffin for immunohistochemical staining. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Amplification Total RNA was extracted from tissues by the acid guanidinium thiocyanate–phenol– chloroform method. Synthesis of the first-strand cDNA was performed with 1 mg of total RNA using a commercially available kit (T-primed First-strand Kit, Pharmacia-LKB, Uppsala, Sweden). One microliter of cDNA was amplified with Taq polymerase (Takara, Otsu, Japan) in a total volume of 50 ml containing 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 100 mM of each dNTP, and 50 pmol of each primer. The primers, K3 (59-GACACATATTGCTTCAATGCTTCAGC) and K4 (59-GATGCCAAGATGATCAGCCATTCTGGAAT), were homologous to the standard region flanking the insertion site of alternatively spliced variants. PCR was performed with 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min using a thermal cycler (GeneAmp PCR 2400, Perkin–Elmer Cetus, Norwalk, CT). Then, 10 ml of PCR products was electrophoresed in a 1.5% agarose gel and transferred to nylon membranes (Hybond N, Amersham, Buckinghamshire, UK) and hybridized with 32P-labeled probe S1 (59-CCTGAAGAAGATTGTACATCAGTCACAGAC) which corresponds to exon 4 and recognizes the constant region. The membranes were also hybridized with 32P-labeled probes M1, M2, and M3, which correspond to V6 (exon 11), V7 (exon 12), and V10 (exon 15), respectively. Following hybridization, the filters were washed and autoradiography was performed. As a positive control, normal uterine cervical epithelium known to contain the mRNA of CD44 standard and variant forms was used for RT-PCR. As a negative control, the reaction mixture without cDNA was amplified under the same conditions as described by Fujita et al. [19].
Immunohistochemistry All tissues were fixed in formaldehyde and embedded in paraffin. Tissue sections were cut at 3-m m thickness, mounted on slides, deparaffinized, and rehydrated. Slides were then incubated for 30 min in 0.3% H2O2 in methanol to inactivate endogenous peroxidase activity, and placed in 10% zinc sulfate in distilled water and heated with a microwaves twice for 5 min to retrieve antigenicity. After incubation for 20 min with diluted normal serum to eliminate nonspecific background, slides were covered with monoclonal mouse anti-CD44V antibody diluted 1:1000 with serum/ PBS and incubated for 60 min at room temperature; the antibodies used were VEF7 (anti-CD44 V6) and VEF-9 (anti-CD44 V7) (Bender Medsystems, Vienna, Austria). After a wash in PBS, slides were incubated for 30 min with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA) at room temperature. They were then incubated in peroxidase-conjugated avidin for 30 min and washed with PBS. Color was developed with 0.05% diaminobenzidine and 0.01% H2O2 in PBS (pH 7.6) for 7 min. The nuclei were stained with Mayer’s hematoxylin. A normal cervical tissue that was known to contain the CD44V antigen was used as a positive control slide. As a negative control slide, a normal nonimmune serum was used instead of the primary antiTABLE 2 Clinicopathological Features and Expression of CD44 V6 and V7 in Endometrial Cancera
Variable Stage I II III IV Histological type Adenocarcinoma Adenoacanthoma Adenosqamous carcinoma Clear cell carcinoma Grade 1 2 3 Depth of myometrial invasion ,1/2 $1/2 Lymphatic and vascular invasion Negative Positive Lymph node metastasis Negative Positive a
V6 (1)
V7 (1)
15/16 3/3 6/6 2/2
14/16 3/3 6/6 2/2
20/21 4/4 1/1 1/1
19/21 4/4 1/1 1/1
10/10 5/6 5/5
10/10 5/6 4/6
12/13 14/14
13/13 12/14
13/13 13/14
12/13 13/13
25/26 1/1
25/2 0/1
There was no significant relationship between any parameter and CD44 V6 or V7 expression.
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187
FIG. 1. Immunohistochemical staining of CD44 isoform containing V6 and V7. (A) Normal endometrium stained for CD44 V6. (B) Endometrial carcinoma stained for CD44 V6. (C) Normal endometrium stained for CD44 V7. (D) Endometrial carcinoma stained for CD44 V7.
body. Strong and/or widespread staining was regarded as positive and weak and focal staining as negative, as described by Kainz et al. [20]. Results were assessed by three independent observers. RESULTS Expression of CD44 mRNA in Normal Endometrium and Endometrial Cancer Hybridization with S1 probe which recognizes the constant region yielded a major band approximately 482 bp along with several larger bands. This major band, corresponding to CD44 standard form (CD44S), was detected in all normal endometrial samples and endometrial cancers. Hybridization with V6-, V7-, and V10-specific probes yielded up to five bands, from approximately 600 to 1300
bp. The band patterns of V6 and V7 were very similar, suggesting that V6 and V7 were associated. The variant forms containing V6 and V7 exons were expressed in a significantly larger proportion of endometrial cancers (P , 0.01). CD44 V10 was expressed in most normal endometrial samples and endometrial cancers, and showed no significant difference (Table 1). No correlation was found between the expression of CD44 V6 and V7 and any clinicopathological features of endometrial cancer such as stage, histological type, grade, and depth of myometrial invasion (Table 2), and there was no statistical difference between expression of CD44 V6 and V7 and age or menopausal status of normal endometrium and endometrial cancer. There was also no significant correlation between CD44 V10 expression and any clinicopathological feature, age, or menopausal status (data not shown).
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Immunohistochemistry CD44 V6 and V7 proteins were detected in respectively 26% (5/19) and 42% (8/19) of normal endometrial samples, and 48% (13/27) and 61% (16/27) of endometrial cancers. There was no significant difference in expression of CD44 V6 and V7 between normal endometria and endometrial cancer. And no correlation was found with any clinicopathological feature, age, or menstrual status. Only epithelial cells of the endometrial glands and tumor cells of the majority of cancer samples were stained; to the contrary, stromal cells and myometrium were not stained at all (Fig. 1). DISCUSSION Since it was reported that a splice variant of CD44 was closely related to metastatic potential, CD44V has been demonstrated in many types of human cancers. Overexpression of CD44 V6 and V7 has been reported in gastric cancer [21], breast cancer [10], and colorectal cancer [18, 22, 23]. In colon cancer [10, 18] and gastric cancer [13], in particular, overexpression of CD44 V6 and V7 is associated with lymph node metastasis. In addition, in breast cancer [9], non-Hodgkin’s lymphoma [8], and cervical carcinoma [24], the presence of these variants is correlated with poorer overall survival than in patients with variant-negative tumors. To the contrary, in prostate cancer [15, 16] and squamous cell carcinoma of the head and neck [17], expression of CD44V is decreased compared with that in corresponding normal tissues, and reduced expression of CD44V is associated with tumor metastasis. Thus the clinical significance of CD44V remains to be determined. In this study, we found that CD44S is expressed in all normal endometria and endometrial cancers, and CD44 V6 and V7 are expressed in significantly higher proportions in cancer than in normal endometrial samples. CD44S is expressed in many types of epithelial cells. Furthermore, in many types of cancer such as breast cancer, colon cancer, and cervical cancer, CD44S is expressed in all samples of tumor. These findings suggest that CD44S may play a role in cell– cell and cell– matrix interaction in cancerous tissues as well as normal epithelium. Since most samples of cancer tissues expressed these variants (V6 in 93%, V7 in 96%), we were unable to determine the correlation between the expression of V6 and V7 and lymph node metastasis or prognosis, respectively. The variant forms containing V6 and V7 were expressed in significantly higher proportions of endometrial cancer than in normal endometria as assessed by RT-PCR. However, these variants were also expressed in 58 – 63% of normal endometria. These data suggest that CD44 V6 and V7 may play a role in the function of normal endometrium. The dysregulation of the expression in cancer cells resulted in the increase in variant expression. The previous paper [19] reported that expression of both CD44S and CD44V was reduced in endometrial cancer and that the absence of CD44 was related to a high incidence of
lymph-vascular space involvement of endometrial cancer. The reason for the difference from our findings was unclear. It may be due to differences in the probes used for hybridization or in the experimental conditions. In this study, we used both the RT-PCR method and immunohistochemical staining to detect CD44 V6 and V7. While a high frequency of CD44 V6 and V7 expression was detected by RT-PCR, the corresponding proteins were detected in relatively few cases by immunohistochemical staining. The discrepancy in frequency of detection of CD44V between RTPCR and immunohistochemistry is due to the difference in the sensitivities of the experimental methods used [25]. In conclusion, we have shown that expression of CD44 V6 and V7 is significantly increased in endometrial cancer compared with normal endometria. However, overexpression of CD44V is not related to metastatic potential in endometrial cancer. REFERENCES 1. Zaino RJ, Kurman R, Herbold D, Gliedman J, Bundy BN, Voet R, Advani H: The significance of squamous differentiation in endometrial carcinoma: Data from a Gynecologic Oncology Group study. Cancer 68:2293–2302, 1991 2. Jalkanen S, Bargatze RF, Toyos J, Butcher EC: Lymphocyte recognition of high endothelium: Antibodies to distinct epitopes of an 85–95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells. J Cell Biol 105:983–990, 1987 3. Heider KH, Hofmann M, Hors E, Berg F, Ponta H, Herrlich P, Pals ST: A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps. J Cell Biol 120:227–233, 1993 4. Fox SB, Fawcett J, Jackson DG, Collins I, Gatter KC, Harris AL, Gearing A, Simmons DL: Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms. Cancer Res 54:4539 – 4546, 1994 5. Gunthert U: CD44: A multitude of isoforms with diverse functions. Curr Top Microbiol Immunol 184:47– 63, 1993 6. Gunthert U, Hofmann M, Rudy W, Reber S, Zoller M, Haussmann I, Matzku S, Wenzel A, Ponta H, Herrlich P: A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65:13–24, 1991 7. Koopman G, Heider KH, Horst E, Adolf GR, Berg F, Ponta H, Herrlich P, Pals ST: Activated human lymphocytes and aggressive non-Hodgkin’s lymphomas express a homologue of the rat metastasis-associated variant of CD44. J Exp Med 177:897–904, 1993 8. Ristamaki R, Joensuu H, Soderstrom KO, Jalkanen S: CD44v6 expression in non-Hodgkin’s lymphoma: An association with low histological grade and poor prognosis. J Pathol 176:259 –267, 1995 9. Kaufmann M, Heider KH, Sinn HP, Minckwitz G, Ponta H, Herrlich P: CD44 variant exon epitopes in primary breast cancer and length of survival [see comments]. Lancet 345:615– 619, 1995 10. Rodriguez C, Monges G, Rouanet P, Dutrillaux B, Lefrancois D, Theillet C: CD44 expression patterns in breast and colon tumors: A PCR-based study of splice variants. Int J Cancer 64:347–354, 1995 11. Yamaguchi A, Urano T, Goi T, Saito M, Takeuchi K, Hirose K, Nakagawara G, Shiku H, Furukawa K: Expression of a CD44 variant containing exons 8 to 10 is a useful independent factor for the prediction of prognosis in colorectal cancer patients. J Clin Oncol 14:1122–1127, 1996
CD44 VARIANTS IN ENDOMETRIAL CANCER 12. Yamaguchi A, Saito M, Gio T, Iida A, Takeuchi K, Hirose K, Nakagawara G, Urano T, Furukawa K, Shiku H: Expression of CD44 variant exons 8 –10 in gastric cancer. Jpn J Cancer Res 86:1166 –1171, 1995 13. Dammrich J, Vollmers HP, Heider KH, Muller HH: Importance of different CD44v6 expression in human gastric intestinal and diffuse type cancers for metastatic lymphogenic spreading. J Mol Med 73:395– 401, 1995 14. Iida N, Bourguignon LY: New CD44 splice variants associated with human breast cancers. J Cell Physiol 162:127–183, 1995 15. Kallakury BV, Yang F, Figge J, Smith KE, Kausik SJ, Tacy NJ, Fisher HA, Kaufman R, Figge H, Ross JS: Decreased levels of CD44 protein and mRNA in prostate carcinoma: Correlation with tumor grade and ploidy. Cancer 78:1461–1469, 1996 16. Nagabhushan M, Pretlow TG, Guo YJ, Amini SB, Pretlow TP, Sy MS: Altered expression of CD44 in human prostate cancer during progression. Am J Clin Pathol 106:647– 651, 1996 17. Herold MC, Seiter S, Born AI, Patzelt E, Schupp M, Zoller J, Bosch FX, Zoller M: Expression of CD44 splice variants in squamous epithelia and squamous cell carcinomas of the head and neck. J Pathol 179:66 –73, 1996 18. Wielenga VJ, Heider KH, Offerhaus GJ, Adolf GR, Berg F, Ponta H, Herrlich P, Pals ST: Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression. Cancer Res 53:4754 – 4756, 1993 19. Fujita N, Yaegashi N, Ide Y, Sato S, Nakamura M, Ishiwata I, Yajima A: Expression of CD44 in normal human versus tumor endometrial
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tissues: Possible implication of reduced expression of CD44 in lymphvascular space involvement of cancer cells. Cancer Res 54:3922–3928, 1994 20. Kainz C, Kohlberger P, Sliutz G, Tempfer C, Heinzl H, Reinthaller A, Breitenecker G, Koelbl H: Splice variants of CD44 in human cervical cancer stage IB to IIB. Gynecol Oncol 57:383–387, 1995 21. Yokozaki H, Ito R, Nakayama H, Kuniyasu H, Taniyama K, Tahara E: Expression of CD44 abnormal transcripts in human gastric carcinomas. Cancer Lett 83:229 –234, 1994 22. Imazeki F, Yokosuka O, Yamaguchi T, Ohto M, Isono K, Omata M: Expression of variant CD44-messenger RNA in colorectal adenocarcinomas and adenomatous polyps in humans. Gastroenterology 110:362–368, 1996 23. Yoshida K, Sugino T, Bolodeoku J, Warren BF, Goodison S, Woodman A, Toge T, Tahara E, Tarin D: Detection of exfoliated carcinoma cells in colonic luminal washings by identification of deranged patterns of expression of the CD44 gene. J Clin Pathol 49:300 –305, 1996 24. Kainz C, Kohlberger P, Tempfer C, Sliutz G, Gitsch G, Reinthaller A, Breitenecker G: Prognostic value of CD44 splice variants in human stage III cervical cancer. Eur J Cancer 31:1706 –1709, 1995 25. Dall P, Heider KH, Sinn HP, Skroch AP, Adolf G, Kaufmann M, Herrlich P, Ponta H: Comparison of immunohistochemistry and RT-PCR for detection of CD44v-expression, a new prognostic factor in human breast cancer. Int J Cancer 60:471– 477, 1991
71, 185–189 (1998)
GO985169
Overexpression of CD44 Variants 6 and 7 in Human Endometrial Cancer Masumi Katsura, M.D.,1 Hiroyuki Furumoto, M.D., Masato Nishimura, M.D., Masaharu Kamada, M.D., and Toshihiro Aono, M.D. Department of Obstetrics and Gynecology, University of Tokushima School of Medicine, 3-18-15 Kuramoto, Tokushima 770, Japan Received April 1, 1998
CD44 is broadly expressed on many types of epithelial cells as well as on lymphocytes [3, 4]. There are at least 17 variant isoforms (CD44V) generated by alternative splicing of at least 10 exons [5]. A variant form of CD44 has recently been found to affect metastatic potential of cancer cells. Gunthert et al. [6] reported that CD44V containing V4 –V7 were expressed only in metastatic cell lines, and not in nonmetastatic cell lines. Furthermore, overexpression of this variant isoform is sufficient for the nonmetastasizing rat pancreatic carcinoma cells to acquire metastatic potential. CD44V expression has been demonstrated in many types of human cancers. Expression of CD44V is increased in non-Hodgkin’s lymphoma [7, 8], breast cancer [9], colon cancer [10, 11], and gastric cancer [12, 13] compared with corresponding normal tissues. Furthermore, CD44V expression has been correlated with lymph node metastasis in colon cancer [10], gastric cancer [12, 13], and breast cancer [14]. On the other hand, in prostate cancer [15, 16] and squamous cell carcinoma of the head and neck [17], expression of CD44V is decreased compared with corresponding normal tissues, and reduced CD44V expression is associated with tumor metastasis. The marked differences in CD44V expression by histological type suggest that the functions of CD44V may differ by histological type. Indeed, from the clinical point of view, several papers have reported a correlation between metastatic potential or prognosis and CD44 V6, V7, and V10 [10, 18]. However, very little is known about the expression of CD44V in endometrial cancer. In this study, we examined the expression of CD44 V6, V7, and V10 in normal endometrium and endometrial cancer.
Objectives. The expression of CD44 V6, V7, and V10 in normal endometrium and endometrial cancer was compared. Methods. Using reverse transcription polymerase chain reaction (RT-PCR) blot analysis, the expression of mRNA containing CD44 V6, V7, and V10 was determined in 19 normal endometrium and 27 endometrial cancer samples. Immunohistochemical staining of CD44 V6 and V7 was performed in the same samples. Results. In RT-PCR analysis, the CD44 variant forms containing V6 and V7 exons were expressed in 96 and 93% of endometrial cancer tissues, respectively. These proportions were significantly higher than those in normal endometrium (V6, 63%; V7, 58%) (P < 0.01). CD44 V10 was expressed in 96% of endometrial cancers and 89% of normal endometrial samples. In immunohistochemical staining, CD44 V6 and V7 were detected in 48 and 61% of endometrial cancers and in 26 and 42% of normal endometrial samples, respectively. Neither of these differences was significant. No correlation was found between the expression of CD44 variants and any clinicopathological features. Conclusion. CD44 V6 and V7 were expressed in a significantly larger proportion of endometrial cancers than normal endometrial samples. However, they were also expressed in a considerable proportion of normal endometria. These findings suggest that CD44 V6 and V7 play roles in normal endometrial function and overexpression of CD44 V6 and V7 is not related to the metastatic potential of endometrial cancer. © 1998 Academic Press Key Words: CD44 variants; endometrial cancer; endometrium; reverse transcription polymerase chain reaction; immunohistochemistry.
INTRODUCTION Stage I and II endometrial cancer has been shown to have a good clinical course. However, if extrauterine metastases occur, the prognosis is very poor. Although degree of myometrial invasion and histological grade have been reported as predictors of metastasis [1], an additional predictor of metastasis would aid in the clinical management of patients with endometrial cancer. CD44 is a cell surface adhesion molecule that was originally reported as a lymphocyte homing receptor [2]. Moreover, 1
To whom reprint requests should be addressed. Fax: 81– 886 –31–2630. E-mail: [email protected]. 185
MATERIALS AND METHODS Tumor and Normal Tissue Samples Nineteen normal endometrial tissue samples (ages 27–76; 10 premenopausal, 9 postmenopausal) and twenty-seven samples of endometrial carcinoma (ages 28 –76; 1 premenopausal, 26 postmenopausal) were obtained from patients at surgery. The 27 samples comprised 21 adenocarcinomas, 4 adenoacanthomas, 1 adenosquamous carcinoma, and 1 clear cell carcinoma. 0090-8258/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Expression of CD44S and CD44V in Normal Endometrium and Endometrial Cancer
Normal endometrium Premenopausal Postmenopausal Endometrial cancer Premenopausal Postmenopausal a
CD44S (%)
V6 (%)
V7 (%)
V10 (%)
19/19 (100) 9/9 10/10 27/27 (100) 1/1 26/26
12/19 (63) 6/9 6/10 26/27 (96)a 1/1 25/26
11/19 (58) 6/9 5/10 25/27 (93)a 1/1 24/26
17/19 (89) 8/9 9/10 26/27 (96) 1/1 25/26
P , 0.01, Fisher’s exact test, compared with normal endometrium.
All samples were frozen in liquid nitrogen immediately after resection and stored at 280°C until use. Adjacent sections were fixed in paraformaldehyde and embedded in paraffin for immunohistochemical staining. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Amplification Total RNA was extracted from tissues by the acid guanidinium thiocyanate–phenol– chloroform method. Synthesis of the first-strand cDNA was performed with 1 mg of total RNA using a commercially available kit (T-primed First-strand Kit, Pharmacia-LKB, Uppsala, Sweden). One microliter of cDNA was amplified with Taq polymerase (Takara, Otsu, Japan) in a total volume of 50 ml containing 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 100 mM of each dNTP, and 50 pmol of each primer. The primers, K3 (59-GACACATATTGCTTCAATGCTTCAGC) and K4 (59-GATGCCAAGATGATCAGCCATTCTGGAAT), were homologous to the standard region flanking the insertion site of alternatively spliced variants. PCR was performed with 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min using a thermal cycler (GeneAmp PCR 2400, Perkin–Elmer Cetus, Norwalk, CT). Then, 10 ml of PCR products was electrophoresed in a 1.5% agarose gel and transferred to nylon membranes (Hybond N, Amersham, Buckinghamshire, UK) and hybridized with 32P-labeled probe S1 (59-CCTGAAGAAGATTGTACATCAGTCACAGAC) which corresponds to exon 4 and recognizes the constant region. The membranes were also hybridized with 32P-labeled probes M1, M2, and M3, which correspond to V6 (exon 11), V7 (exon 12), and V10 (exon 15), respectively. Following hybridization, the filters were washed and autoradiography was performed. As a positive control, normal uterine cervical epithelium known to contain the mRNA of CD44 standard and variant forms was used for RT-PCR. As a negative control, the reaction mixture without cDNA was amplified under the same conditions as described by Fujita et al. [19].
Immunohistochemistry All tissues were fixed in formaldehyde and embedded in paraffin. Tissue sections were cut at 3-m m thickness, mounted on slides, deparaffinized, and rehydrated. Slides were then incubated for 30 min in 0.3% H2O2 in methanol to inactivate endogenous peroxidase activity, and placed in 10% zinc sulfate in distilled water and heated with a microwaves twice for 5 min to retrieve antigenicity. After incubation for 20 min with diluted normal serum to eliminate nonspecific background, slides were covered with monoclonal mouse anti-CD44V antibody diluted 1:1000 with serum/ PBS and incubated for 60 min at room temperature; the antibodies used were VEF7 (anti-CD44 V6) and VEF-9 (anti-CD44 V7) (Bender Medsystems, Vienna, Austria). After a wash in PBS, slides were incubated for 30 min with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA) at room temperature. They were then incubated in peroxidase-conjugated avidin for 30 min and washed with PBS. Color was developed with 0.05% diaminobenzidine and 0.01% H2O2 in PBS (pH 7.6) for 7 min. The nuclei were stained with Mayer’s hematoxylin. A normal cervical tissue that was known to contain the CD44V antigen was used as a positive control slide. As a negative control slide, a normal nonimmune serum was used instead of the primary antiTABLE 2 Clinicopathological Features and Expression of CD44 V6 and V7 in Endometrial Cancera
Variable Stage I II III IV Histological type Adenocarcinoma Adenoacanthoma Adenosqamous carcinoma Clear cell carcinoma Grade 1 2 3 Depth of myometrial invasion ,1/2 $1/2 Lymphatic and vascular invasion Negative Positive Lymph node metastasis Negative Positive a
V6 (1)
V7 (1)
15/16 3/3 6/6 2/2
14/16 3/3 6/6 2/2
20/21 4/4 1/1 1/1
19/21 4/4 1/1 1/1
10/10 5/6 5/5
10/10 5/6 4/6
12/13 14/14
13/13 12/14
13/13 13/14
12/13 13/13
25/26 1/1
25/2 0/1
There was no significant relationship between any parameter and CD44 V6 or V7 expression.
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187
FIG. 1. Immunohistochemical staining of CD44 isoform containing V6 and V7. (A) Normal endometrium stained for CD44 V6. (B) Endometrial carcinoma stained for CD44 V6. (C) Normal endometrium stained for CD44 V7. (D) Endometrial carcinoma stained for CD44 V7.
body. Strong and/or widespread staining was regarded as positive and weak and focal staining as negative, as described by Kainz et al. [20]. Results were assessed by three independent observers. RESULTS Expression of CD44 mRNA in Normal Endometrium and Endometrial Cancer Hybridization with S1 probe which recognizes the constant region yielded a major band approximately 482 bp along with several larger bands. This major band, corresponding to CD44 standard form (CD44S), was detected in all normal endometrial samples and endometrial cancers. Hybridization with V6-, V7-, and V10-specific probes yielded up to five bands, from approximately 600 to 1300
bp. The band patterns of V6 and V7 were very similar, suggesting that V6 and V7 were associated. The variant forms containing V6 and V7 exons were expressed in a significantly larger proportion of endometrial cancers (P , 0.01). CD44 V10 was expressed in most normal endometrial samples and endometrial cancers, and showed no significant difference (Table 1). No correlation was found between the expression of CD44 V6 and V7 and any clinicopathological features of endometrial cancer such as stage, histological type, grade, and depth of myometrial invasion (Table 2), and there was no statistical difference between expression of CD44 V6 and V7 and age or menopausal status of normal endometrium and endometrial cancer. There was also no significant correlation between CD44 V10 expression and any clinicopathological feature, age, or menopausal status (data not shown).
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Immunohistochemistry CD44 V6 and V7 proteins were detected in respectively 26% (5/19) and 42% (8/19) of normal endometrial samples, and 48% (13/27) and 61% (16/27) of endometrial cancers. There was no significant difference in expression of CD44 V6 and V7 between normal endometria and endometrial cancer. And no correlation was found with any clinicopathological feature, age, or menstrual status. Only epithelial cells of the endometrial glands and tumor cells of the majority of cancer samples were stained; to the contrary, stromal cells and myometrium were not stained at all (Fig. 1). DISCUSSION Since it was reported that a splice variant of CD44 was closely related to metastatic potential, CD44V has been demonstrated in many types of human cancers. Overexpression of CD44 V6 and V7 has been reported in gastric cancer [21], breast cancer [10], and colorectal cancer [18, 22, 23]. In colon cancer [10, 18] and gastric cancer [13], in particular, overexpression of CD44 V6 and V7 is associated with lymph node metastasis. In addition, in breast cancer [9], non-Hodgkin’s lymphoma [8], and cervical carcinoma [24], the presence of these variants is correlated with poorer overall survival than in patients with variant-negative tumors. To the contrary, in prostate cancer [15, 16] and squamous cell carcinoma of the head and neck [17], expression of CD44V is decreased compared with that in corresponding normal tissues, and reduced expression of CD44V is associated with tumor metastasis. Thus the clinical significance of CD44V remains to be determined. In this study, we found that CD44S is expressed in all normal endometria and endometrial cancers, and CD44 V6 and V7 are expressed in significantly higher proportions in cancer than in normal endometrial samples. CD44S is expressed in many types of epithelial cells. Furthermore, in many types of cancer such as breast cancer, colon cancer, and cervical cancer, CD44S is expressed in all samples of tumor. These findings suggest that CD44S may play a role in cell– cell and cell– matrix interaction in cancerous tissues as well as normal epithelium. Since most samples of cancer tissues expressed these variants (V6 in 93%, V7 in 96%), we were unable to determine the correlation between the expression of V6 and V7 and lymph node metastasis or prognosis, respectively. The variant forms containing V6 and V7 were expressed in significantly higher proportions of endometrial cancer than in normal endometria as assessed by RT-PCR. However, these variants were also expressed in 58 – 63% of normal endometria. These data suggest that CD44 V6 and V7 may play a role in the function of normal endometrium. The dysregulation of the expression in cancer cells resulted in the increase in variant expression. The previous paper [19] reported that expression of both CD44S and CD44V was reduced in endometrial cancer and that the absence of CD44 was related to a high incidence of
lymph-vascular space involvement of endometrial cancer. The reason for the difference from our findings was unclear. It may be due to differences in the probes used for hybridization or in the experimental conditions. In this study, we used both the RT-PCR method and immunohistochemical staining to detect CD44 V6 and V7. While a high frequency of CD44 V6 and V7 expression was detected by RT-PCR, the corresponding proteins were detected in relatively few cases by immunohistochemical staining. The discrepancy in frequency of detection of CD44V between RTPCR and immunohistochemistry is due to the difference in the sensitivities of the experimental methods used [25]. In conclusion, we have shown that expression of CD44 V6 and V7 is significantly increased in endometrial cancer compared with normal endometria. However, overexpression of CD44V is not related to metastatic potential in endometrial cancer. REFERENCES 1. Zaino RJ, Kurman R, Herbold D, Gliedman J, Bundy BN, Voet R, Advani H: The significance of squamous differentiation in endometrial carcinoma: Data from a Gynecologic Oncology Group study. Cancer 68:2293–2302, 1991 2. Jalkanen S, Bargatze RF, Toyos J, Butcher EC: Lymphocyte recognition of high endothelium: Antibodies to distinct epitopes of an 85–95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells. J Cell Biol 105:983–990, 1987 3. Heider KH, Hofmann M, Hors E, Berg F, Ponta H, Herrlich P, Pals ST: A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps. J Cell Biol 120:227–233, 1993 4. Fox SB, Fawcett J, Jackson DG, Collins I, Gatter KC, Harris AL, Gearing A, Simmons DL: Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms. Cancer Res 54:4539 – 4546, 1994 5. Gunthert U: CD44: A multitude of isoforms with diverse functions. Curr Top Microbiol Immunol 184:47– 63, 1993 6. Gunthert U, Hofmann M, Rudy W, Reber S, Zoller M, Haussmann I, Matzku S, Wenzel A, Ponta H, Herrlich P: A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65:13–24, 1991 7. Koopman G, Heider KH, Horst E, Adolf GR, Berg F, Ponta H, Herrlich P, Pals ST: Activated human lymphocytes and aggressive non-Hodgkin’s lymphomas express a homologue of the rat metastasis-associated variant of CD44. J Exp Med 177:897–904, 1993 8. Ristamaki R, Joensuu H, Soderstrom KO, Jalkanen S: CD44v6 expression in non-Hodgkin’s lymphoma: An association with low histological grade and poor prognosis. J Pathol 176:259 –267, 1995 9. Kaufmann M, Heider KH, Sinn HP, Minckwitz G, Ponta H, Herrlich P: CD44 variant exon epitopes in primary breast cancer and length of survival [see comments]. Lancet 345:615– 619, 1995 10. Rodriguez C, Monges G, Rouanet P, Dutrillaux B, Lefrancois D, Theillet C: CD44 expression patterns in breast and colon tumors: A PCR-based study of splice variants. Int J Cancer 64:347–354, 1995 11. Yamaguchi A, Urano T, Goi T, Saito M, Takeuchi K, Hirose K, Nakagawara G, Shiku H, Furukawa K: Expression of a CD44 variant containing exons 8 to 10 is a useful independent factor for the prediction of prognosis in colorectal cancer patients. J Clin Oncol 14:1122–1127, 1996
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