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CD44 glycoproteins in colorectal cancer; expression, function and prognostic
value
Wielenga, V.J.M.
Publication date
1999
Link to publication
Citation for published version (APA):
Wielenga, V. J. M. (1999). CD44 glycoproteins in colorectal cancer; expression, function and
prognostic value.
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Chapter 4
CD44 SPLICE VARIANTS AS PROGNOSTIC
MARKERS IN COLORECTAL CANCER.
Vera J.M. Wielenga, Robbert van der Voort, Jan-Willem R. Mulder, Philip
M. Kruyt, Wiebo F. Weidema, Johan Oosting , Cees A. Seldenrijk, Cees van
Krimpen, G. Johan A. Offerhaus, Steven T. Pais
Scan J Gastroenterol 1998, 33:82-87
CD44 Splice Variants as Prognostic Markers in Colorectal Cancer
V. J. M. WIELENGA, R. VAN DER VOORT, J. W. R. MULDER, P. M. KRUYT,
W. F. WEIDEMA, J. OUSTING, C. A. SELDENR1JK, C. VAN KRIMPEN, G. J. A. OFFERHAUS & S. T. PALS
Depts. of Pathology, Surgery, and Clinical Epidemiology and Biostatistics, Academic Medical Center, University of Amsterdam, Amsterdam, and Depts. of Surgery and Pathology, Reinier de Graaf Hospital, Delft, The Netherlands
Wielenga VJM, van der Voort R, Mulder JWR, Kruyt PM, Weidema WF, Oosting J, Seldenrijk CA, van Krimpen C, Offerhaus GJA, Pals ST. CD44 splice variants as prognostic markers in colorectal cancer. Scand J Gastroenterol 1998;33:82-87.
Background: Splice variants of CD44 play a causal role in the metastatic spread of pancreatic carcinoma in the rat. In previous studies we have shown that homologues of these CD44 isoforms (CD44v6) are overexpressed during colorectal tumorigenesis in man and that CD44v6 overexpression is associated with an unfavorable prognosis in this disease. In the present study we have assessed the prognostic significance of CD44 variants containing exon v5. In addition, we have used a panel of different antibodies against CD44v6 and applied a combined scoring system to improve its value as prognosticator. Methods: Expression of CD44 variants was studied by immunohistochemistry on frozen tissue sections, and the prognostic value of the CD44 variant expression was assessed using univariate and multivariate analysis. Results: Our studies show that expression of CD44v6, but not CD44v5, has significant prognostic value. Analysis of CD44v6 expression by means of a combined scoring system, on the basis of a panel of three different monoclonal antibodies (mAbs), makes CD44v6 a highly significant prognostic marker that is independent of Dukes stage, tumor grade, or tumor localization. Conclusion: Assessment of CD44v6 expression by a combination of mAbs yields an independent prognosticator that may be of value in identifying patients with a high propensity to develop distant metastasis.
Key words: CD44; cell adhesion; colorectal cancer; metastasis; prognosis
S. T. Pals, M.D., Ph.D., Dept. of Pathology, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (fax: +31-20-6960389)
Tumor metastasis is the principal cause of death in cancer patients. Therefore, effective assessment of the metastatic potential of tumors is a prime goal for cancer research (1). In patients with colorectal cancer approximately half of the carcinomas that appear to be radically excised at surgery (Dukes B and C) will eventually give rise to progressive disease due to the presence of occult metastases at the time of operation. Markers that can predict metastasis in these tumor stages are of great potential value for the clinician, since they identify patients who are likely to benefit from adjuvant chemotherapy and/or radiotherapy. Several recent studies suggest that CD44 splice variants might be such markers.
CD44 is a widely distributed family of cell surface glycoproteins which has been implicated in several important biologic processes including hematopoiesis (2, 3), lympho-cyte activation (2,4, 5, 6), lympholympho-cyte homing (2, 7), and tumor progression and metastasis (2, 8-19). Due to alternative splicing of at least lOexons (vl-vlO), which encode domains of the extracellular portion of the molecule, a large number of CD44 splice variants is generated (11,20). Posttranslational modification, which includes N- and O-linked glycosylation and decoration with glycosaminoglycan (GAG) side chains, adds further diversity to CD44 (2,21,22). The finding that CD44 variants, containing sequences encoded by exon v6.
confer full metastatic potential to rat carcinoma cell lines (11) led to the hypothesis that homologous CD44 variants might also mediate metastasis formation in humans.
We have previously shown that CD44 splice variants containing exon v5 and v6, which are hardly or not at all expressed in the normal epithelium of the colon and rectum (23,24), are increasingly overexpressed during colorectal tumorigenesis (14, 15). Furthermore, we found evidence of an association between overexpression of CD44v6-containing splice variants and risk of tumor-related death (16). In the present study we have extended these observations by exploring the prognostic significance of CD44 variants containing exon v5. Furthermore, we show that assessment of CD44v6 expression by a panel of monoclonal antibodies (mAbs) makes CD44v6 a highly significant marker for metastatic propensity in colorectal cancer, independent of conventional prognosticators.
MATERIALS AND METHODS
Tissue samples
The study set consisted of 68 primary colorectal carcino-mas, removed at operation between 1 January 1983 and 1 January 1 1986 at the Dept. of Surgery, Reinier de Graaf
B
| | v 2 | v3 | v4 | v5 | v6 | v7 |v8 | v9 [ vlO
S
VFF7Fig. IA. Schematic representation of the CD44 gene. Open boxes indicate exons that can be alternatively spliced. TM = transmembrane region. 1B. Schematic representation of the CD44 protein with localizations of the epitopes that are recognized by the monoclonal antibodies VFF8, VFF4, VFF7, and VFF18. Anti-variant antibodies were raised against a bacterially expressed fusion protein encoded by pGEX CD44v HPKII (v3-vl0). v2-10 are domains encoded by variant exons.
Hospital, Delft, for which snap-frozen tissue and follow-up until 1 June 1992 (6.5-9.5 years) was available. The mean age of the patients at diagnosis was 69.7 years (range, 39-92 years), and the male to female ratio was 28:40. The tumors were staged in accordance with a modification of the original Dukes classification (25). The four Dukes classes are defined as follows: Dukes A, disease limited to the bowel wall; Dukes B, extension through the deep muscle without metastases; Dukes C, regional lymph-node metastases but radical resec-tion; Dukes D, distant metastases. The tumors were graded as well-differentiated, moderately differentiated, or poorly differentiated, using standard criteria (26). Right-sided tumors came from the cecum, ascending colon, and transverse colon, and left-sided tumors from the descending colon, sigmoid, and rectum.
Detection of CD44 variants
Frozen tissue sections were tested for the expression of CD44 splice variants by immunohistochemistry as described previously (6, 15), using a panel of mAbs raised against a bacterially expressed fusion protein encoded by exons v3~ vlO. Tonsillar tissue was used as a positive control in each staining, and isotype (IgGl)-matched mAbs were used as negative controls. The mAbs used were VFF4, VFF7, and VFF18, which recognize CD44v6 (Bender Co., Vienna, Austria), and VFF8, which recognizes CD44v5 (Bender Co.) (Fig. 1). A detailed description of these antibodies and their specificities has been published elsewhere (6, 23). The anti-CD44v6 mAb VFF18 recognizes the epitope QWFGNRWHEGYRQT on exon v6 (23). The other two anti-CD44v6 mAbs compete with VFF18 and with each other for binding, hence their epitopes are identical or overlapping. The relative affinities of VFF18, VFF4, and VFF7, as determined by BlAcore using as immobilized antigen the
bacterial GST fusion protein CD44v3-10, were 100%, 71%, and 15%, respectively (27). All slides were read by four independent observers (J. W. Mulder, C. A. Seldenrijk, G. J. A. Offerhaus, S. T.Pals), and discrepancies were solved by consensus. During the whole procedure the observers were blinded for disease outcome. On the basis of the estimated percentage of the positively staining tumor cells, the stained samples were scored as follows: 0 (low/negative) = less than 10% of the cells positive; 1 (intermediate) = 10% to 50% of the cells positive; and 2 (high) = more than 50% of the cells positive. For the three mAbs that recognize exon v6 the scores of each patient were added. Hence, the possible 'cumulative score' for exon v6 expression for each patient ranges from 0 to 6. For statistical analysis patients were grouped into three categories: CD44v6 low = 0-2; CD44v6 intermediate = 3 and 4;CD44v6high = 5 and 6.
Statistical analysis
In univariate analysis survival functions were estimated with the Kaplan-Meier method. Comparison of survivor functions between groups was performed with the log-rank test. Multivariate analysis by means of the Cox regression model was executed with the expression of exon v6 'cumu-lative score', tumor localization, tumor stage, and tumor
Table I. Percentage of colorectal carcinomas with low, and high staining for CD44 variants
intermediate, Expression level VFF8 (v5), n = 68 VFF7 (v6), n = 68 VFF4 (v6), n = 68 VFF18(v6), n = 68 Low Intermediate High 18% 29% 5 3 % 8 1 % 12% 7% 3 1 % 3 1 % 38% 6% 2 1 % 73%
Low, intermediate, and high: expression on <10%, 10-50%, and >50% of the tumor cells, respectively.
Table II. Prognostic significance of tumor localization, Dukes stage, tumor grade, and CD44 variant expression
Univariate analysis No. of patients Median survival (days) P valuet Tumor localization Right colon 24 256 0.17 Left colon 44 1150 Tumor stage Dukes B 27 2681 <0.0001 Dukes C 32 899 Dukes D 9 59 Tumor grade Well differentiated 11 896 0.02 Moderately differentiated 44 1150 Poorly differentiated 13 211 Exon v6* Low 26 2160 0.0005 Intermediate 31 997 High 11 61 Exon v5 Low 12 1577 0.5 Intermediate 20 1098 High 36 950
* Cumulative score after staining with monoclonal antibodies VFF4, VFF7, and VFF18 (see Materials and Methods).
t Log-rank test.
grade as variables. With the aid of the stepwise backward regression technique, the most parsimonious model was achieved. The likelihood ratio test was used to test for significance. The P values of the variables were calculated by eliminating each variable.
RESULTS
Expression of CD44 variants in colorectal cancer The expression of CD44 splice variants in the study group of 68 colorectal carcinomas is shown in Table I. Each of the variants examined was expressed at levels that varied greatly between individual tumors: whereas some tumors were completely negative for a given variant, other tumors expressed the same variant strongly on all or virtually all of the tumor cells.
Positivity (intermediate or high expression) of CD44v5 was present in 82%. For CD44v6 the percentage of positive tumors varied greatly depending on the mAb used for the detection of v6: With mAbs VFF7, VFF4, and VFF18, CD44v6 expression was found in 19%, 69%, and 94% of the carcinomas, respectively (Table I). These differences in the percentage of positive tumors corresponded consistently with differences in antibody affinity (VFF18 > VFF4 > VFF7). Nevertheless, the staining patterns obtained with the three different mAbs were identical.
CD44 variants and survival
To test the hypothesis that CD44 splice variant expression plays a role in the dissemination and prognosis of colorectal
EXON V6 i'"'L. (VFF4) Ui '•"-1 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 9 TIME (YEARS)
Fig. 2. Kaplan-Meier curves showing the relation between Dukes stage, CD44v5 and v6 expression, and survival in patients with colorectal carcinoma, measured with the individual monoclonal antibodies: VFF8 </> = 0.5), VFF4 (/» = 0.004), VFF7 (/» = 0.002), and VFF18 (P = 0.01). Dotted line = low expression; broken line = intermediate expression; unbroken line = high expression; + = censored cases. The upper right diagram shows the relation between Dukes stage and survival. Dotted line = Dukes B; broken line = Dukes C; unbroken line = Dukes D; + = censored cases.
cancer, we assessed the relation between CD44v5 and CD44v6 expression and patient survival in our study group (Table O, Fig. 2). Additional risk factors considered in the study were primary tumor localization (right- versus left-sided), Dukes stage, and tumor grade. Univariate analysis showed that CD44v6 but not CD44v5 expression was correlated with survival. For CD44v6 a significant correlation was found with all three mAbs used. Fig. 3 shows the cumulative score for CD44v6, obtained by integrating the scores from the three separate mAbs. In addition to CD44v6, Dukes stage (Table II, Fig. 2) and tumor grade (Table O) were also correlated with tumor-related death. CD44v6 expression was significantly correlated with Dukes stage (chi-square, P = 0.04), and there was no significant correlation between CD44v6 expression and tumor grade (chi-square, P = 0.3).
CD44 splice variants as prognostic parameter
" I t+"H™
0 1 2 3 4 5 6 7 8 9 TIME (YEARS)
Fig. 3. Kaplan-Meier curve showing the relation between CD44v6 expression and survival after integrating the scores of the three separate monoclonal antibodies (VFF7, VFF4, and VFF18) against CD44v6 (P = 0.0005).
independent prognostic value, multivariate analysis was performed with the CD44v6 cumulative score, tumor stage, tumor grade, and tumor localization as variables (Table III). In tumor stage only patients with Dukes B and C tumors were included, representing the patients who underwent radical surgery. After stepwise backward regression, Dukes stage and CD44v6 expression were the determining factors in the assessment of prognosis (Table Til). In Fig. 4 the final effect of exon v6 is shown versus an average Dukes classification, visualizing the clinical impact of CD44v6 expression on survival.
DISCUSSION
Prediction of disease outcome in colorectal cancer is currently
based mainly on the stage of tumor growth at the time of resection. However, patients with tumors of the same stage often show a diametrically different outcome. Hence, addi-tional independent prognostic markers are needed to provide a rationale to adjust therapeutic approaches. In our prr.ent study we provide evidence that CD44 splice variants have this potential.
Using the same patient group for our study material, we had previously found that expression of CD44v6 on the resected primary tumor is associated with an unfavorable prognosis in colorectal carcinoma (16). In this study we have further explored and improved the value of CD44v6 as a prognos-ticator in colorectal cancer, by assessing CD44v6 expression with a panel of three mAbs, which recognize identical or overlapping epitopes on CD44v6 but differ in affinity (23, 27). This affinity difference was reflected by the sensi-tivity with which CD44v6 was detected and resulted in a vastly different number of tumors stained positive with each of the antibodies (Table I). A significant correlation between CD44v6 expression and survival was observed with each of the individual mAbs (Fig. 2). However, by integrating the results of the mAb panel to a cumulative CD44v6 score (Fig. 3), which represents a semiquantitative assessement of CD44v6 expression, a more optimal differentiation between risk groups was obtained. In multivariate analysis of Dukes B and C tumor patients the patient group that underwent radical surgery and hence might benefit from adjuvant therapy for occult metastatic disease, this cumulative CD44v6 score emerged as a highly significant independent prognostic factor (Table in. Fig. 4).
The correlation of CD44v5 expression with patient survival was not statistically significant but showed the same trend as CD44v6 (Table II, Fig. 2).
Several studies addressing the expression of CD44 splice variants in colorectal cancer have recently been published (12-16,28-33). They indicate that CD44 splice variants
Table III. Independent prognostic significance of tumor stage, tumor grade, tumor localization, and CD44v6 expression
Full model After backwards stepwise regression
Hazard ratio Hazard ratio
Standard (exponent Standard (exponent
Multivariate analysis p1-coefficient error coefficient) P value ^-coefficient error coefficient) P value Tumor stage Dukes B Dukes C 0.9449 0.3654 2.5725 0.008 1.0379 0.3511 2.8232 0.002 Tumor grade Well-differentiated Moderately differentiated -0.7237 0.6087 0.4850 Poorly differentiated -0.7509 0.4104 0.4719 0.21 Tumor localization Left colon Right colon -0.2761 0.3944 0.7587 0.48 Exon v6* Low 0.5788 0.3754 1.7840 0.5341 0.3544 1.7060 High 1.4907 0.5691 4.4403 0.04 1.4726 0.5180 4.3608 0.02
* Cumulative score after staining with monoclonal antibodies VFF4, VFF7, and VFF18 (see Materials and Methods).
63
* 0.8
fc~.
Xv;--.
> 0.6 < =• 0.4—|
v ' •
2t ,
U 0.2 1 0 1 2 3 4 5 6 7 8 9 TIME (YEARS)Fig. 4. Impact of CD44v6 expression on survival in Dukes B and C patients. Dotted line = low expression; broken line = intermediate expression; unbroken line = high expression (P = 0.02).
b e c o m e o v e r e x p r e s s e d during colorectal tumorigenesis. T h i s overexpression p r e s u m a b l y represents an early event in the a d e n o m a - c a r c i n o m a s e q u e n c e , since it is already detectable in a d e n o m a s ( 1 4 , 1 5 , 2 8 - 3 0 , 3 2 , 3 3 ) , w h e r e it m a y occur before K-ras and p 5 3 g e n e mutation (30). In e a c h t u m o r a c o m p l e x splice pattern with several different splice variants c o n t a i n i n g various c o m b i n a t i o n s of e x o n products e n c o d e d by v 2 v l 0 is present ( 1 2 1 6 , 2 8 , 2 9 , 3 3 ) . T h e precise c o m p o s i -tion of these different variants and their func-tions in tumori-genesis are still u n k o w n . A b n o r m a l expression of variant e x o n s w a s also recently s h o w n in exfoliated colorectal c a n c e r cells, suggesting that it m a y be helpful in the early, n o n -invasive detection of colorectal cancer (34).
In conclusion, our current findings provide further e v i d e n c e of a role of C D 4 4 splice variants in colorectal t u m o r i g e n e s i s and of their utility as prognostic markers. Specifically, the data s h o w that i m m u n o h i s t o c h e m i c a l assessement of C D 4 4 splice variant expression with a panel of three m A b s against C D 4 4 v 6 , which could easily be performed on a routine basis, yields an independent prognosticator that might be used to identify patients with a high propensity to develop distant metastasis. T h e s e patients m i g h t benefit from adjuvant c h e m o t h e r a p y and/or radiotherapy.
A C K N O W L E D G E M E N T S
W e thank Dr. G. R. Adolf and Dr. E. Patzelt for m o n o c l o n a l antibodies V F F 4 , V F F 7 , V F F 8 , and V F F 1 8 and M r . A. Pronk for expert technical assistance.
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Received 19 June 1997 Accepted 17 October 1997
