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Short Communications |
From the Division of Cell and Molecular
Pathology*
and the Department of Pathology and
Urologic Clinic,
University Hospital,
University of Zürich, Zürich; the Institute for
Pathology
and Urologic
Clinics,§
Cantonal Hospital St. Gallen, St.
Gallen; the Institute for Pathology¶
and
Urologic Clinics,||
City Hospital Triemli, Zürich;
the Urologic Clinics,**
Limmattal Hospital,
Schlieren; the Clara
Hospital,
Basel; and the Urologic
Clinics
and Institute for
Pathology,§§
University of Basel,
Basel, Switzerland
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD44 glycoproteins are promising candidates, because the CD44 variant glycoprotein containing sequences encoded by exon 6 has been shown to confer metastatic potential on rat pancreatic carcinoma cells6,7 and to be implicated in progression and metastasis formation in various human malignancies.8 The transitional epithelium of urinary bladder and cell lines derived from it, as well as cell lines from TCCs, have been shown to express various CD44 isoforms including CD44v3, -v4, -v5, and -v9.9,10 In addition, retention of certain intronic sequences has been reported to occur frequently in TCC but hardly ever in normal transitional epithelium.11 Recent studies have shown that CD44v6 expression is reduced in poorly differentiated, invasive TCCs, as compared to well or moderately differentiated noninvasive tumors.12-14 Furthermore, a correlation between CD44 expression and prognosis has been reported.13 In long-term follow-up, strong expression of CD44v6 was related to high survival probability and independently related to favorable outcome in muscle invasive tumors, whereas standard CD44 was an independent prognostic factor in superficial tumors. Other investigators, however, failed to establish a prognostic value for CD44v6 for tumor recurrence and patient survival.15
Based on our previously obtained data on a small collection of superficial TCCs,16 we have evaluated the value of CD44 immunohistochemistry in predicting recurrence in 241 superficial (pTa and pT1) TCCs from patients for whom long-term follow-up data were available. Here we show that focal loss of immunostaining, observed with monoclonal antibodies against CD44v3 and -v6 protein, is a significant prognostic factor that helps to identify patients at high risk for recurrence of noninvasive (pTa) TCCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Formalin-fixed, paraffin-embedded tissue samples of primary TCCs of the urinary bladder were available from the archives of the Institute of Pathology at the University of Basel, the Cantonal Hospital St. Gallen, and the Triemli Hospital in Zurich, Switzerland. The series consisted of 241 superficial bladder carcinomas (pTa and pT1). Tumor stage and grade were defined according to UICC and WHO classifications.17,18 One pathologist (G. S.) reviewed all slides of all tumors. Stage pT1 was defined by the presence of both unequivocal tumor invasion of the suburothelial stroma and tumor-free fragments of the muscular bladder wall. Biopsies containing carcinoma with stroma invasion, but lacking fragments of the muscular bladder wall, were excluded, as were true papillomas (grade 0). Of the 241 cases of superficial TCCs, 167 (69.3%) were pTa and 74 (30.7%) were pT1; 65 (27.4%) were G1, 127 (52.7%) were G2, and 49 (20.3%) were G3.
Clinical histories of patients (194 males, 47 females) with superficial bladder cancer were retrospectively evaluated by reviewing the patients’ charts and contacting the attending physicians. The average age of these patients was 65.6 years (range, 20–92 years). Regular follow-up cystoscopies had been performed at least at 3, 9, and 15 months, then annually until the endpoint of this study (recurrence, progression, or last control). Information on the number of tumors present in the bladder was obtained for 172 patients (75 multicentric, 97 unicentric). The medium follow-up period was 54 months (range, 6–167 months). Intravesical treatment had been performed in 60 patients (mitomycin in 29, BCG in 26, epirubicin in 2, fenorubicin in 2, adriblastin in 1, tigason in 1, and tumosteron in 1 patient). Recurrences were defined as cystoscopically visible tumors. Tumor progression was defined as the presence of muscle invasion (stage pT2 or higher) in a subsequent biopsy. An increase in stage from pTa to pT1 was not defined as progression because not all subsequent biopsies of our patients were available for review. Such a review is mandatory for studies investigating pTa-to-pT1 progression because of the interobserver variability among different pathologists, ranging from 25 to 40%, for the distinction of pTa and pT1.4,5
Immunohistochemistry
CD44 protein expression was analyzed immunohistochemically, employing protein A-purified mouse monoclonal IgG reactive with CD44 protein isoforms containing human variant exons 3 (clone 3G5) and human variant exons 6 (clone 2F10) purchased from R&D Systems (Abingdon, UK). Immunostaining for CD44 was performed on paraffin sections, applying microwave antigen retrieval as previously described.19 After quenching of endogenous peroxidase activity (0.6% H2O2 in absolute methanol, 15 minutes) and section conditioning20 with 1.5% normal horse serum and 4% fat-free milk in isotonic Tris-HCl buffer, pH 6.5, for 20 minutes, sections were incubated with primary antibodies (1 µg/ml) for 60 minutes. Following buffer rinses, sections were processed by a streptavidin-biotin-peroxidase technique according to the manufacturer’s recommendations (Jackson ImmunoResearch Laboratories, West Grove, PA). Peroxidase activity was revealed according to Adams.21
Specificity controls included the omission of primary and/or secondary antibodies as negative controls and the use of normal skin as positive controls.
Evaluation of Immunostaining and Statistical Analysis
During the whole immunohistochemical evaluation, the observers
were blinded for disease outcome. A first evaluation scheme assessed
the CD44-positive tumor cut surface area regardless of the staining
intensity. Tumors were grouped into those with 
50% and those with
<50% positive tumor cut surface. Furthermore, focal loss of
immunostaining for both CCD44v3 and -v6 was evaluated in serial
sections. Focal loss of immunostaining was defined as the appearance of
variably sized regions of the urothelium lacking immunoreactivity with
the monoclonal antibodies throughout its entire thickness. The second
scheme evaluated the dominant pattern of immunostaining. Homogenous
staining was defined as extending evenly through the entire thickness
of the neoplastic urothelium, whereas restricted immunostaining was
defined as extending through less than 3/4 of the thickness.
Relationships between variables obtained in form of frequency tables
were statistically analyzed using the 
2
test.22
Survival curves regarding tumor recurrence were
obtained according to Kaplan-Meier and the significance of their
differences was assessed by the log-rank test. Hazard ratios indicating
the relative risk of recurrence of one group compared with the other
were calculated. A Cox proportional hazard model was used to
determine the parameters, with greatest influence on the risk of
recurrence and progression.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transitional epithelium adjacent to tumors exhibited
immunolabeling in the basal cell layer, decreasing in intensity toward
the surface, with the most superficial layers of the urothelium
including the umbrella cells being unlabeled, as reported by
others.10,12,13
In the 241 TCCs investigated, two
prevailing staining patterns were observed: restricted staining
extending through less than 3/4 of the urothelium thickness and
homogenous staining extending evenly through the entire thickness of
the neoplastic urothelium (Figure 1)
.
Tumors exhibiting either of these labeling patterns could present
variably sized regions with loss of immunostaining (Figure 1)
. Such a
focal loss of immunostaining was strongly associated with advanced
tumor stage and high histological grade. A focal loss of immunostaining
was found in 89 of 167 pTa (53.3%), 66 of 74 pT1 (89.2%;
P < 0.0001 for pTa vs. pT1), 28 of 65 G1
(43.1%), 79 of 127 G2 (62.2%), and 48 of 49 G3 tumors (98.0%,
P < 0.0001, three-group contingency table analysis for
grade).
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In our collective, 129 of 241 (53.5%) patients suffered from
recurrent disease. To determine the predictive value of CD44
immunohistochemistry for recurrence of superficial TCCs, the initial
biopsies of all pTa and pT1 tumor patients, with and without recurrent
disease, were analyzed. Among the parameters analyzed, a significant
difference was observed only in association with focal loss of
immunostaining for both CD44v3 and -v6 protein. The group of pTa/pT1
TCCs exhibiting unlabeled areas for both CD44v3 and -v6, irrespective
of their size (50% or <50% positive tumor cut surface), had a
significantly shorter recurrence-free interval
(P = 0.0132, Figure 2a
) and was at significantly higher risk
of developing tumor recurrence (hazard ratio 1.26, confidence limits
1.05–1.53). None of the other analyzed staining patterns distinguished
tumors with recurrence from those without (data not shown). In
addition, several subgroups of the patient collective were analyzed
regarding the association between focal loss of immunostaining and
recurrence. Although patients with noninvasive pTa tumors showed a
significant difference in recurrence-free survival
(P = 0.005, hazard ratio 1.34, confidence limits
1.09–1.67, Figure 2b
), the group of minimally invasive pT1 tumors did
not (P = 0.864). The association with recurrence
was preserved in the subgroup of patients with pTa tumors who had not
received chemotherapy (P = 0.031). The group of
pTa TCCs with chemotherapy was too small for statistical analysis
(n = 33). Multicentricity
(P = 0.008), but not grade
(P = 0.750, Figure 2c
) or stage
(P = 0.689), was linked to an increased risk of
tumor recurrences in univariate analysis. A multivariate analysis of
our collective, including grade (P = 0.58),
stage (P = 0.50), multicentricity
(P = 0.013), and focal loss of immunostaining
(P = 0.063), revealed that only multicentricity
(P = 0.013) was an independent predictor of
recurrences, whereas focal loss of immunostaining
(P = 0.063) did not reach the level of
significance.
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) and, to a lesser extent,
multicentricity (P = 0.050) and focal loss of
immunostaining (P = 0.055), were related to
tumor progression in univariate analysis, but not the histological
grade (P = 0.301). In multivariate analysis,
only stage (P < 0.0001) and multicentricity
(P = 0.014) were linked to subsequent tumor
progression, not grade (P = 0.645) or focal loss
of immunostaining (P = 0.141). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that focal loss of CD44v3 and -v6 immunostaining in the
biopsies proved to be of predictive value for recurrence in superficial
TCCs. Although the loss of immunostaining varied in size, variation of
this parameter was not of additional significance, because TCCs
exhibiting 50% behaved similarly to those with <50% unstained
tumor cut surface area. Focal loss of immunostaining occurred in the
great majority of invasive and in half of noninvasive tumors. Although
related to recurrence in the noninvasive group, CD44 was not related to
progression in the two groups. Because focal CD44 loss occurred in most
tumors, it is unlikely to be a causal event in progression. It will be
interesting to determine how focal loss of CD44 immunostaining is
related to other prognostic factors in superficial bladder cancer. It
must be emphasized that our study was performed on routinely
formaldhyde-fixed and paraffin-embedded tumor tissue using a standard
antigen retrieval technique,23
commercially available
reagents, and a standard immunohistochemical procedure. Thus, detection
of focal loss of immunostaining for CD44v3 and -v6 is easily
practicable in surgical pathology laboratories. We observed differences
between noninvasive (pTa) and minimally invasive (pT1) tumors. Patients
with noninvasive TCCs displaying areas with loss of labeling for both
CD44v3 and -v6 showed a significantly shorter recurrence-free survival
time, whereas no such correlation could be established for minimally
invasive (pT1) tumors. These findings are consistent with other marked
genetic differences found between pTa and pT1 TCCs.24,25
From the present results it can be concluded that loss of
immunostaining for CD44v3 and -v6 protein is an event occurring early
during progression of TCC and represents a useful prognostic factor for
recurrence. It could be speculated that CD44v3- and -v6-negative tumor
areas might consist of an expanding subpopulation of tumor cells with
more aggressive growth properties.
In agreement with previously published data on CD44 expression,12-14,26 an inverse correlation existed between the expression of CD44v3 and -v6 proteins and histological grade, as well as tumor stage. Similar observations were made for carcinomas derived from other stratified epithelia, such as squamous cell carcinomas of the skin,19 and for breast carcinoma.27 Thus, reduced expression of CD44 variant forms may have prognostic value in certain carcinoma types, whereas in others, including renal cell28 and colorectal carcinomas,29 overexpression seems to be positively correlated with tumor differentiation and to have prognostic value.
From the present findings arises the question whether the altered CD44
protein expression and tumor recurrence in TCCs are causally related.
We recognize that immunohistochemistry neither permits
direct studies of the complex splice patterns present in carcinomas,
including TCC,9
nor reveals the composition of these
different variants and their functions. One might speculate that
the absence of larger CD44 variant molecules at the cell surface may
facilitate cell detachment and favor interaction of shorter CD44
molecules with the extracellular matrix to permit migration and
invasion, as shown for melanoma cell
lines.30,31
It is also possible that the changed CD44
expression represents solely an epiphenomenon of dedifferentiation,
which occurs in parallel to processes that cause tumor recurrence and
invasion in TCCs. Indeed, a simultaneously occurring decrease
in the expression of CD44v6, E-cadherin,12,15
and
6ß4
integrin32
could be demonstrated in invasive TCC.
In conclusion, our current findings provide further evidence of the involvement of CD44 splice variants in the tumorigenesis of TCC. In terms of diagnosis and practicability, they show that detection of focal loss of immunohistochemical staining for CD44v3- and -v6-containing isoforms may provide an additional parameter in identifying patients with TCC at risk for tumor recurrence and that this information can be obtained on formalin-fixed, paraffin-embedded tissues. This immunohistochemical evaluation may be especially useful because the extent of loss of staining does not seem to be important and the identification of one area with unequivocal loss of immunostaining may be sufficient to define an increased risk for recurrence in affected patients.
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Acknowledgements |
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Footnotes |
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Supported by the Swiss National Science Foundation and the Canton of Zürich. V. T. was recipient of a special educational grant from the University Hospital Zürich.
Accepted for publication July 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6ß4 integrin and collagen VII in bladder cancer. Am J Pathol 1994, 144:787-795[Abstract]
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