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Japanese Journal of Clinical Oncology Pages 609-614

Ratio of CD44 Epithelial Variant to CD44 Hematopoietic Variant is a Useful Prognostic Indicator in Gastric and Colorectal Carcinoma
Introduction
Materials and Methods
   Patients
   Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
   Statistics
Results
   CD44 Expression in Gastric Carcinoma
   CD44 Expression in Colorectal Carcinoma
Discussion
References
Ratio of CD44 Epithelial Variant to CD44 Hematopoietic Variant is a Useful Prognostic Indicator in Gastric and Colorectal Carcinoma

Ratio of CD44 Epithelial Variant to CD44 Hematopoietic Variant is a Useful Prognostic Indicator in Gastric and Colorectal Carcinoma

Yuichi Shibuya1, Takahiro Okabayashi2, Kenji Oda1 and Noriaki Tanaka1

1First Department of Surgery, Okayama University Medical School, Okayama and 2Department of Surgery, Kochi Municipal Central Hospital, Kochi, Japan

Background: The CD44 is a cell adhesion molecule that is present as numerous isoforms created by mRNA alternative splicing. Expression of variant isoforms of CD44 is associated with tumor growth and metastasis. The aim of this study was to evaluate whether CD44 isoform expression is a prognostic factor in gastric and colorectal cancer.
Methods: We performed a polymerase chain reaction analysis following reverse transcriptase treatment for CD44 expression in fresh surgical specimens obtained from 25 colon carcinomas and 30 gastric carcinomas and matched adjacent normal mucosa. We determined the epithelial variant/hematopoietic variant (E/H) ratio (the amount of the CD44 epithelial variant transcript relative to the CD44 hematopoietic variant transcript) in cancer tissues and examined it for correlations with clinicopathological parameters and survival rate.
Results: The E/H ratio in tumor tissue was significantly higher than that in adjacent non-cancerous mucosa. In gastric and colorectal cancer tissues, there was no significant relationship between E/H ratio and clinicopathological parameters. However, patients who died due to tumor recurrence had a higher E/H ratio than surviving patients with colorectal cancer. In gastric cancer, patients with high E/H tumors had a shorter survival time than those with low E/H tumors.
Conclusions: Our results suggest that the E/H ratio is a useful indicator of prognosis in gastric and colorectal carcinoma.

Key words: CD44 - gastric carcinoma - colorectal carcinoma - prognosis

INTRODUCTION

The CD44 is a cell surface adhesion molecule, originally described as a lymph node homing receptor on circulating lymphocytes (1). The gene encoding CD44 is found on chromosome 11p. The CD44 protein exists in numerous isoforms generated from the primary transcript by alternative splicing of 10 exons (v1-v10, 2,3). An isoform lacking all 10 variant exons and termed the hematopoietic form (CD44H) is present on cells of normal epithelial and hematopoietic origin. In contrast, the CD44 alternatively spliced variants are expressed predominantly on cells of tumors. The epithelial variant (CD44E), one of the CD44 variants, contains exons v8-v10. Recently, CD44 has been postulated to have a role in tumor metastasis. First Günthert et al. (4) showed that overexpression of one CD44 variant form, encoded by the cDNA clone pMeta-1, conferred metastatic behavior on a non-metastasizing rat pancreatic carcinoma cell line. Overexpression of the epithelial variant CD44E was found in metastasis of colorectal cancer (5). Other authors have found that tumor progression is linked to overexpression of exon v6 in colon cancer (6). Guo et al. (7) reported that serum CD44 concentrations may be an indicator of tumor burden and metastasis in patients with gastric and colon cancer. However, the function of such CD44 isoforms is largely unknown.

In gastric (8), colorectal (9,10), breast (11) and non-Hodgkin's lymphoma (12), several studies employing immunohistochemical methods have indicated that CD44 variant isoforms correlate with length of survival, but no studies have been done using molecular biology techniques. In this study, using reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated the E/H ratio (the amount of CD44E transcript relative to CD44H transcript) in surgical specimens of human gastric and colorectal cancers and matched adjacent normal mucosa. We considered whether the E/H ratio in cancer tissues is a useful indicator of prognosis.

Table 1. Details of patients
  Gastric cancer Colorectal cancer
No. of patients 30 25
Mean age (years) 64 (40-86) 67 (47-88)
Clinical stage Ia 1 I 7
Ib 3 II 5
II 5 IIa 4
IIIa 6 IIIb 5
IIIb 4 IV 4
IVa 0    
IVb 11    

MATERIALS AND METHODS

Patients

Twenty-five patients with colorectal carcinoma had primary surgery in Okayama University Hospital between January 1992 and January 1994 and 30 patients with gastric carcinoma had operations between November 1991 and November 1993. Normal and tumor tissue samples obtained from the surgical resections of these patients (Table 1) were immediately frozen and stored at -80°C. Tissue samples taken from areas more than 5 cm distal to the main tumors are hereafter referred to as normal tissue samples.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted by the acid guanidinum-thiocyanate-phenol-chloroform method (13) and reverse transcripted with oligo dT primers, using MoMuLV reverse transcriptase, for 60 min at 37°C (14). PCR was carried out in a reaction volume of 25 µl containing CD44 primers at 94°C for 30 s, 55°C for 60 s and 72°C for 2 min for 30 cycles in a thermal cycler (Perkin-Elmer/Cetus, Emeryville, CA) (15). The primers used were 5[prime]-CCAATGCCTTTGATGGACCA-3[prime] (sense) and 5[prime]-TGTGAGTGTCCATCTGATTC-3[prime] (antisense). Some resulting bands of RT-PCR were identified by Southern blotting, using the CD44 probe, as being the cloned fragment described above. PCR amplification products were resolved by 7.5% polyacrylamide gel electrophoresis (PAGE) and stained with ethidium bromide. The total intensity of each resulting band was measured with a densitometer (Scanning Imager 300SX; Molecular Dynamics, Sunnyvale, CA). The E/H ratio was represented by the ratio of the numerical value of CD44E of CD44H.

Statistics

The chi-squared test was used where appropriate. The relationship between E/H ratio and tumor tissues and normal tissues was assessed by the Wilcoxon signed rank test. E/H ratios were compared with the clinicopathological features classified according to the General Rules for Gastric Cancer Study of the Japanese Research Society for Gastric Cancer (16) and the General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus of the Japanese Research Society for Cancer of Colon and Rectum (17). Statistical analysis was performed with the t-test. The survival analysis was performed using the Kaplan-Meier method and the generalized Wilcoxon test. P-values of <0.05 were considered statistically significant.

RESULTS

Because primers amplify across the site in the mRNA at which extra segments may be inserted to produce different forms of CD44, the intervening `standard' portion of the molecule will be amplified along with any transcripts from exons 1-10 spliced into it. The amplified fragment will be 334 bp if a sample expresses standard (hematopoietic) CD44 (CD44H) only, a fragment of 730 bp will be produced if it is the epithelial form of CD44 (CD44E) and there will be several other fragments if a sample contains alternatively spliced transcripts (Fig. 1). These appear as bands of differing molecular size when resolved by gel electrophoresis and Southern hybridization with CD44 probes. PCR produced several amplification products (Fig. 2) and Southern blot analysis confirmed that all the bands corresponded to CD44 isoforms.


Figure 1. Schematic representation of the CD44 gene and CD44 protein.


Figure 2. PCR amplification of CD44 cDNA and GAPDH cDNA from surgical specimens representing normal mucosa (N) and colon carcinoma (T).

CD44 Expression in Gastric Carcinoma

CD44H was observed in all tumor tissues and adjacent non-cancerous mucosa, whereas CD44E was detected in 69% (20 of 29) of the tumor tissues and in 40% (12 of 30) of adjacent non-cancerous mucosa. The difference between the two groups was significant (P < 0.05). Other splice variant forms were observed in 38% (11 of 29) of the tumor tissues and in 13% (4 of 30) of non-cancerous mucosa. The average E/H ratios in gastric carcinoma and adjacent non-cancerous mucosa were 0.28 ± 0.16 and 0.17 ± 0.11. The E/H ratio in tumor tissues was significantly higher than in adjacent non-cancerous mucosa (P < 0.01, Wilcoxon signed rank test) (Fig. 3). There was no significant relationship between E/H ratio and histological type, depth of invasion, lymph node metastasis, blood vessel invasion, lymphatic vessel invasion, liver metastasis or peritoneal dissemination (Table 2). Tumors with an E/H ratio of [ge]0.28 were designated high E/H tumors and those with a ratio of <0.28 as low E/H tumors. The correlation between E/H ratio and recurrence and tumor-induced mortality in 14 curatively resected patients was examined. In high E/H tumors there were one patient with Stage Ia disease, two with Stage Ib, two with Stage II, three with Stage IIIa and two with Stage IIIb. In low E/H tumors there was one patient with Stage Ib disease, one with Stage II, one with Stage IIIa and one with Stage IIIb. There was no significant difference between two groups in clinical stages. During follow-up of 3-47 months (mean 29 months), seven of 10 patients with high E/H tumors had local recurrence or distant metastasis and died, whereas none of four patients with low E/H tumors did. The mean E/H ratios in gastric tumors from patients who died due to recurrence was 0.41 ± 0.15, whereas that in surviving patients was 0.24 ± 0.20. The difference between the two groups was not significant (P = 0.09) (Table 3). The Kaplan-Meier curves show that patients with high E/H tumors had a shorter survival time than those with low E/H tumors (P < 0.05) (Fig. 4).


Figure 3. E/H ratio in non-tumor and tumor tissues. Lines connect paired specimens obtained simultaneously from same patient.


Figure 4. Kaplan-Meier analysis of overall survival in patients suffering from gastric carcinoma with low or high E/H ratio.

Table 2. Correlation between E/H ratio and clinicopathological parameters in gastric carcinoma
  E/H ratio Significance
Depth of invasion t1, t2 n = 12 0.33 ± 0.20 N.S.
t3, t4 n = 17 0.25 ± 0.14
L.N. metastasis n0, n1 n = 14 0.26 ± 0.16 N.S.
n2, n3 n = 15 0.29 ± 0.17
Lymphatic vessel invasion (+) n = 24 0.26 ± 0.17 N.S.
(-) n = 4 0.33 ± 0.21
Venous invasion (+) n = 17 0.26 ± 0.19 N.S.
(-) n = 11 0.28 ± 0.14
Liver metastasis (+) n = 2 0.07 ± 0.07 N.S.
(-) n = 27 0.30 ± 0.16
Clinical stage Ia, Ib ,II n = 9 0.31 ± 0.17 N.S.
IIIa, IIIb, IVa, IVb n = 20 0.27 ± 0.17

Table 3. Difference of E/H ratio between surviving patients and dead patients
Carcinoma Patients E/H ratio Significance
Gastric Surviving 0.24 ± 0.20 P = 0.09
Dead 0.41 ± 0.15
Colorectal Surviving 0.57 ± 0.29 P < 0.05
Dead 1.21 ± 0.58

CD44 Expression in Colorectal Carcinoma

The RT-PCR analysis demonstrated CD44H in all tumor tissues and adjacent non-cancerous mucosa. The band of CD44E was detected in 83% (19 of 23) of the tumor tissues and in 37% (seven of 19) of adjacent non-cancerous mucosa, suggesting that the CD44E expression was predominantly observed in colorectal carcinomas compared to normal tissues (P < 0.05, [chi]2 test). Other splice variant forms were observed in 44% (10 of 23) of the tumor tissues and in 26% (five of 19) of non-cancerous mucosa. The average E/H ratios in colorectal carcinoma and adjacent non-cancerous mucosa were 0.66 ± 0.48 and 0.25 ± 0.22, respectively. The E/H ratio in tumor tissues was significantly higher than in adjacent non-cancerous mucosa (P < 0.01, Wilcoxon signed rank test) (Fig. 3). There was no significant relationship between E/H ratio and histological type, depth of invasion, lymph node metastasis, blood vessel invasion, lymphatic vessel invasion, liver metastasis or clinical stage (Table 4). Tumors with an E/H ratio of [ge]0.66 were designated high E/H tumors and those with a ratio of <0.66 as low E/H tumors. The correlation between E/H ratio and recurrence and tumor-induced mortality was examined in 16 curatively resected patients. In high E/H tumors there was one patient with Stage I disease, three with Stage II and two with Stage IIIa. In low E/H tumors there were three patients with Stage I disease, three with Stage II, two with Stage IIIa and two with Stage IIIb. There was no significant difference between two groups in clinical stages. During follow-up of 9-50 months (mean 37 months), three of six patients with high E/H tumors had local recurrence or distant metastasis, compared with only two of 10 patients with low E/H tumors. Tumor-induced mortality was more frequent in high E/H ratio tumors (2/6 vs 0/10, P = 0.05). The mean E/H ratio of tumors from patients who died due to tumor recurrence was 1.21 ± 0.58, whereas that of tumors from surviving patients was 0.57 ± 0.29. The difference between these two groups was significant (P < 0.05) (Table 3). The Kaplan-Meier curves show that patients with high E/H tumors had a shorter survival time than those with low E/H tumors (P = 0.06) (Fig. 5).

Table 4. Correlation between E/H ratio and clinicopathological features in colorectal carcinoma
  E/H ratio Significance
Depth of invasion sm, mp n = 8 0.49 ± 0.26 N.S.
ss, s, si n = 14 1.07 ± 1.02
L.N. metastasis (+) n = 12 0.77 ± 0.57 N.S.
(-) n = 12 0.55 ± 0.35
Lymphatic vessel invasion (+) n = 16 0.57 ± 0.36 N.S.
(-) n = 7 0.67 ± 0.43
Invasion to vein (+) n = 10 0.49 ± 0.38 N.S.
(-) n = 14 0.65 ± 0.39
Liver metastasis (+) n = 2 1.28 ± 0.51 N.S.
(-) n = 23 0.56 ± 0.39
Clinical stage I, II n = 12 0.56 ± 0.32 N.S.
IIIa, IIIb, IV n = 10 1.12 ± 1.16

DISCUSSION

At the time of operation we cannot predict perfectly the prognosis of operated patients by clinicopathological features alone. However, predictions may be made more accurately by investigating the expression of oncogene, tumor-suppressor gene and adhesion molecules which influence tumor growth and metastasis. Several groups have postulated that CD44 variant isoforms correlate with length of survival employing immunohistochemical methods. There have been no studies indicating that CD44 variant isoforms correlate with length of survival using molecular biology techniques. RT-PCR amplification resolved by PAGE can perform quantitative analysis, but immunohistochemical studies cannot. In this study we investigated whether the expression of CD44 variants influences tumor metastasis and can be a prognostic factor in gastric and colorectal carcinomas using RT-PCR.


Figure 5. Kaplan-Meier analysis of overall survival in patients suffering from colorectal carcinoma with low or high E/H ratio.

Tanabe et al. (5) analyzed CD44 splice variants in specimens from normal colonic mucosa, primary colorectal cancers, normal liver and liver metastasis and suggested that overexpression of CD44R1 (CD44E) variant may increase the metastatic potential of those cancers. Yamaguchi et al. (18) analyzed the expression of CD44v8-10 immunohistochemically using the anti-CD44v8-10 monoclonal antibody and determined that it is a useful independent factor for prognosis in colorectal cancer. Tanabe et al. (19) reported that when CD44H was reintroduced into colon carcinoma cells, their in vitro and in vivo growth was significantly reduced. Miwa et al. (20) reported that the difference between E/H ratios of tumor tissue and adjacent non-cancerous mucosa correlated with lymph node metastasis, depth of invasion, blood vessel invasion and lymphatic vessel invasion in gastric carcinoma. We considered that a reduction of CD44H and an increase of CD44E may increase tumor growth rate and metastatic potential and CD44E/H ratio may be a prognostic factor. Using RT-PCR, we amplified CD44 obtained from human gastric and colorectal carcinoma, calculated the E/H ratio in cancer tissues and investigated the correlation between E/H ratio and clinicopathological parameters and survival. No significant association was found between E/H ratio and clinicopathological parameters and clinical stage. These findings differ from other authors' conclusions (20,21). In our studies, patients with liver metastases had higher E/H ratios than patients without liver metastases in colorectal carcinoma, but there was no significant difference. This may be due to the small number of patients with liver metastases in this study. After curative surgery, patients with high E/H ratios had poor prognosis due to tumor recurrence in colorectal and gastric carcinomas despite there being no significant difference in clinical stage between high and low E/H tumors. In gastric carcinoma multivariate analyses showed that the CD44 E/H ratio was not an independent prognostic factor (data not shown). In colorectal carcinoma multivariate analyses could not performed owing to the small number of patients who died. A larger study will help to clarify the independent prognostic significance of the E/H ratio. In univariate analyses the E/H ratio is a useful indicator in the prediction of the prognosis of patients with gastric and colorectal carcinomas and in designing treatment regimens.

References

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2. Jalkanen S, Jalkanen M, Bargatze R, Tammi M, Butcher EC. Biochemical properties of glycoproteins involved in lymphocyte recognition of high endothelial venules in man. J Immunol 1988;141:1615-23. MEDLINE Abstract

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5. Tanabe KK, Ellis LM, Saya H. Expression of CD44R1 adhesion molecule in colon carcinomas and metastases. Lancet 1993;341:725-6. MEDLINE Abstract

6. Wielenga VJ, Heider KH, Offerhaus GJ, Adolf GR, van den Berg FM, Ponta H, et al. Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression. Cancer Res 1993;53:4754-6. MEDLINE Abstract

7. Guo YJ, Liu G, Wang X, Jin D, Wu M, Ma J, Sy MS. Potential use of soluble CD44 in serum as indicator of tumor burden and metastasis in patients with gastric or colon cancer. Cancer Res 1994;54:422-6. MEDLINE Abstract

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19. Tanabe KK, Stamenkovic I, Cutler M, Takahashi K. Restoration of CD44H expression in colon carcinomas reduces tumorigenicity. Ann Surg 1995;222:493-503. MEDLINE Abstract

20. Miwa T, Watanabe A, Yamada Y, Shino Y, Yamada T, Yamashita J, et al. Progression in gastric carcinoma relative to the ratio of CD44 epithelial variant transcript to CD44 hematopoietic variant transcript. Cancer 1996;77:25-9. MEDLINE Abstract

21. Takeuchi K, Yamaguchi A, Urano T, Goi T, Nakagawara G, Shiku H. Expression of CD44 variant exons 8-10 in colorectal cancer and its relationship to metastasis. Jpn J Cancer Res 1995;86:292-7. MEDLINE Abstract

Received April 6, 1998; accepted June 29, 1998
For reprints and all correspondence; Yuichi Shibuya, First Department of Surgery, Okayama University Medical School, 2-5-1, Shikata-cho, Okayama 700-0914, Japan
Abbreviations: mRNA, messenger ribonucleic acid; CD44E, CD44 epithelial variant; CD44H, CD44 hematopoietic variant; RT, reverse transcriptase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis

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Copyright©Japanese Journal of Clinical Oncology, 1998.
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