Int. J. Cancer: 97, 512–517 (2002) © 2002 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
PROGNOSTIC VALUE OF THYMIDINE PHOSPHORYLASE EXPRESSION IN BREAST CARCINOMA Qifeng YANG1,5*, Mattia BARBARESCHI2, Ichiro MORI1, Francesco MAURI1,5, Maurizio MUSCARA` 3, Misa NAKAMURA1, Yasushi NAKAMURA1, Goro YOSHIMURA5, Takeo SAKURAI5, Orazio CAFFO4, Enzo GALLIGIONI4, Paolo DALLA PALMA2 and Kennichi KAKUDO1 1 Second Department of Pathology, Wakayama Medical University School of Medicine, Wakayama City, Japan 2 Department of Histopathology, St. Chiara Hospital, Trento, Italy 3 Department of Histopathology, SS. Trinita` Hospital, Borgomanero, Italy 4 Department of Medical Oncology, St. Chiara Hospital, Trento, Italy 5 Department of Surgery, Affiliated Kihoku Hospital, Wakayama Medical University School of Medicine, Wakayama, Japan Thymidine phosphorylase (TP), also known as plateletderived endothelial cell growth factor (PD-ECGF), is an enzyme that catalyzes the reversible dephosphorylation of thymidine, deoxyuridine and their analogs. TP has also angiogenic properties, although the precise mechanism by which it promotes angiogenesis is not known. We examined TP expression using immunohistochemistry (654-1 Mab) in 182 invasive breast carcinomas (67 N0 and 115 N1/2; median follow-up 78 months [range, 3–177]; 51 patients treated with adjuvant systemic cyclophosphamide, methotrexate and 5-fluorouracil [CMF] chemotherapy and 82 with tamoxifen). High TP expression was found in 142 cases (78%) and correlated with lower histologic grade and low p53 expression. No correlation was found between TP expression and vascular density. TP-positive tumors had a significant increase in both disease-free survival (DFS; p ⴝ 0.0025) and overall survival (OS; p ⴝ 0.0070) in the total cohort of patients and in the subgroups of node-positive patients and patients treated with CMF adjuvant therapy; no significant difference in either DFS or OS was observed in patients without CMF treatment. Our findings suggest that TP has little effect on tumor angiogenesis of breast carcinoma, whereas it could represent an interesting marker that could predict response to CMF chemotherapy. © 2002 Wiley-Liss, Inc. Key words: breast cancer; thymidine phosphorylase; adjuvant therapy;prognosis
Thymidine phosphorylase (TP) is an enzyme specifically involved in the reversible dephosphorylation of thymidine, deoxyuridine and their analogs to their respective bases and 2-deoxy-Dribose-1-phosphate.1 TP is also able to catalyze the conversion of the pyrimidine antimetabolite 5-fluorouracil (5-FU) to 5-fluoro-2⬘deoxyuridine, the first step in 1 of the metabolic activation pathways of this chemotherapeutic agent to deoxyribonucleotides.2 TP has also been identified as an angiogenic factor and identical to platelet derived endothelial cell growth factor (PD-ECGF). 3–5 TP is angiogenic in vivo and stimulates chemotaxis of endothelial cells in vitro.6 – 8 Recently, several antibodies have been generated that allow the immunohistochemical investigation of TP expression.9,10 TP expression has been observed in many normal human tissues: the predominant cells expressing TP are macrophages, but epithelial cells stain as well.9 Many studies have also shown that TP is upregulated in several tumor types,9 including breast carcinomas, where it is expressed by both epithelial and stromal cells including macrophages.11 In intraductal breast carcinomas, epithelial TP expression is correlated with the presence of a dense vascular rim around tumor cell nests, but neither with stromal vascularity nor with relapse-free survival.12 In invasive breast cancer TP is upregulated in 39 –52% of cases,11,13 and its relationship with vascularity is controversial: some authors report that no relationship was found between vascularity and TP expression in either the carcinoma or the inflammatory cells,14 whereas others reported the opposite.11 These conflicting data may reflect the complexity of the angiogenic process and may also suggest that in breast carcino-
genesis TP is important in initial remodeling of the preexisting vascular network but that other factors are needed for extensive induction of vessels growth.12 Preclinical studies have shown a correlation between fluoropyrimidine sensitivity and TP levels. Transfection experiments have provided evidence that TP mediates the sensitivity of HT-29 human colon carcinoma cells to 5-FU15 and of several cell lines (MCF-7 human breast cancer cells, PC-9 lung adenocarcinoma cells, and KB epidermal carcinoma cells) to 5⬘-deoxy-5-fluorouridine (5⬘-DFUR), a prodrug of 5-FU.16 –18 In the clinical setting, it has been suggested that TP may correlate with the efficacy of adjuvant therapy with 5-FU and its derivatives.13,19,20 A recent study has also shown that high expression of TP in cell lines potentiates the effect of the cytotoxic drug methotrexate,21 offering an additional rationale to investigate TP expression in human neoplasms. 5-FU and methotrexate (as well as cyclophosphamide) are used in the CMF treatment regimen of breast cancer. It can be therefore hypothesized that overexpression of TP in breast cancer may increase its sensitivity to CMF chemotherapy, as suggested by the study of Fox et al.20 In the present study we evaluated TP immunohistochemical expression in relation to tumor angiogenesis and its possible role as a prognostic and/or predictive factor in relation to different treatments in 182 Italian breast carcinoma patients with long-term follow-up. MATERIAL AND METHODS
Patients and tumors We investigated 182 consecutive breast carcinoma patients who underwent surgery from January 1984 to August 1991 in Trento, Italy; the study period ended by December 7, 1999. Surgical samples were fixed in buffered formalin and routinely processed. All cases were reviewed by 2 pathologists, and sections from 1 representative paraffin block were selected and sent to Wakayama, Japan for immunohistochemical studies. For statistical purposes, all cases were subdivided into infiltrating ductal carcinoma (IDC; 152 cases) and non-IDC (30 cases) groups. Tumor grading was performed according to the method of Elston and Ellis.22 Seventy cases were node-negative,and 115 were node-positive. The median follow-up duration was 78 (range, 3–177) months. Adjuvant sysThe first two authors contributed equally to this work. *Correspondence to: Second Department of Pathology, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama City 641-0012, Japan. Fax: ⫹81-73-446-4825. E-mail:
[email protected] Received 23 April 2000; Revised 27 July 2001; Accepted 6 August 2001 Published online 22 October 2001; DOI 10.1002/ijc.1633
THYMIDINE PHOSPHORYLASE IN BREAST CANCER
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FIGURE 1 – Thymidine phosphorylase (TP) expression patterns in infiltrating ductal carcinomas: in 1 case the neoplastic cells show diffuse and intense immunostaining in almost all tumor cells (a), and in the other case the tumor cells are completely unreactive with concurrent staining of stromal cells (b).
temic CMF chemotherapy (Milan protocol)23 was given to 51 patients (46 node-positive and 5 node-negative cases). Adjuvant hormonal therapy (tamoxifen, 20 mg daily) was given to 82 patients. Immunohistochemistry All immunostaining was performed using a microwave antigenretrieval system. Sections were stained for TP (anti-TP mouse monoclonal antibody 654-1, diluted 1:1,000; Nippon Roche Research Center, Kanagawa, Japan) at room temperature and processed with a standard streptavidin-biotin complex technique (Dako LSAB system, Dako, Carpinteria, CA).24 A total of 108 tumors were also assessed for tumor vascularity using CD31 (JC70 monoclonal antibody [Mab]; Dako) immunostaining. Microvessel density was evaluated as described by Weidner counting the number of microvessels in the hot-spot areas.25 All tumors were also stained for estrogen receptor (ER; ER1D5 MAb; Dako) and p53 (DO7 MAb; Novocastra, Newcastle-upon-Tyne, UK).26,27 Negative controls were obtained by omitting primary antibodies. The specimens immunostained for TP were evaluated without knowledge of the clinicopathologic features. For our study TP expression was evaluated only in tumor cells and not in stromal macrophages. Tumor TP staining was compared with the surrounding normal epithelium and scored in 3 groups as follows: 1, no staining or ⱖ50% of tumor cells demonstrating weaker staining than that of normal epithelium; 2, ⱖ50% of tumor cells demonstrating similar intensity to that of normal epithelium; and 3, ⱖ50% of tumor cells demonstrating stronger staining than that of normal epithelium.
This scoring system was selected because it seemed reasonably simple and because of the assumed hypothesis that we needed to identify cases with TP expression similar to or higher than the one observed in normal tissue. All samples were evaluated by 3 of our authors. Therefore, for each case there were 3 scores in our database. In case of discordance between the scores reported by the observers, the final score was defined as follows: (i) in case of only 1 discordant score it was the score reported by 2 of the 3 pathologists; and (ii) if all the 3 authors had given 3 different scores, the cases were reevaluated and an agreement was reached. For statistical analysis, tumors were considered TP-positive when they were scored 2 or 3. Cases were considered positive for ER and p53 if reactive cells were ⬎10% and ⬎15%, respectively.27 Statistical analysis Descriptive statistics comparing TP expression with conventional markers of tumor aggressiveness were analyzed by standard chi-square tests, Fisher’s exact test or Mann-Whitney test as appropriate. Estimates of disease-free survival (DFS) and overall survival (OS) were calculated by the Kaplan-Meier product-limit method and the differences assessed by the log rank test. Probabilities of DFS (OS) were calculated from the date of breast carcinoma diagnosis to either the date at which relapse (death) from breast carcinoma was clinically identified or the date of last contact. Multivariate survival analysis using Cox’s proportional hazard regression model was carried out to assess the independent contribution of each variable to DFS and OS. All p-values were 2-tailed and the 0.05 level was considered statistically significant.
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TABLE I – RELATIONSHIP BETWEEN THYMIDINE PHOSPHORYLASE (TP) EXPRESSION AND OTHER PARAMETERS1 Factor
Histology IDC Other Tumor size (cm) ⱕ2 ⬎2 Nodal metastasis Negative Positive Grade 1 and 2 3 ER status Negative Positive p53 Negative Positive MVD (no.)
TP Negative
Positive
p-value
0.6296 35 5
117 25
15 23
75 62
12 28
55 87
12 27
79 60
19 20
45 95
0.1027 0.3570 0.0061 0.0616 0.0094 25 15 56.286
110 23 39.125
0.1119
1
IDC, infiltrating ductal carcinoma; ER, estrogen receptor; MVD, microvessel density.
A computer program package (StatView 5.0, Abacus Concepts, Berkeley, CA) was used for all statistical testing and management of the database. RESULTS
TP tumor cell imunoreactivity was both nuclear and cytoplasmic but occasionally only 1 of these was present. In some cases, nearly all invasive cells were immunopositive for TP (Fig. 1a), whereas in others, the invasive cells were nearly all negative (Fig. 1b). Immunoreactivity was occasionally focal and often more intense at the infiltrating tumor edge. Tumor-associated macrophages were strongly positive. All 3 authors gave the same score for 67% (122/182) of our patients; an agreement between at least 2 observers was reached in all but 2 cases. Because of the impossibility of discussing all cases at a multiheaded microscope, as 1 of the observers was in Japan and 2 in Italy, we decided to use for each case the score obtained by at least 2 observers. The 67% complete interobserver agreement is not very high, and it could be hypothesized that in future studies on TP evaluation one should use a more objective computer-assisted image analyzing system. One hundred forty-three of the (78.6%) cases were regarded as positive for TP. In our series, high TP expression was associated with lower tumor grade (p ⫽ 0.006) and negative p53 status (p ⫽ 0.009), with no significant correlations between TP expression and tumor size, histologic type, nodal status and MVD; a trend was seen for TP-positive cases being associated with positive ER status, but it did not reach statistical significance (p ⫽ 0.06; Mann-Whitney test; Table I). Univariate survival analysis showed statistically significant associations of tumor size, nodal status, grade, ER, p53 and TP (Fig. 2) with DFS (Table II) and OS (Table III). Multivariate analysis showed that tumor size, grade and nodal status were of independent significant prognostic value for DFS; nodal status, grade and ER were of independent significant value for OS (data not shown). We performed further analyses on subgroups of patients adjusted by tumor size, nodal status, ER status and adjuvant therapy (Table IV).High TP expression was associated with prolonged DFS and OS in the subgroup of patients treated with CMF adjuvant therapy (p ⫽ 0.01 and p ⫽ 0.009, respectively; Fig. 3) and in node-positive patients (p ⫽ 0.0343 and p ⫽ 0.0186, respectively); no relation with survival was seen in the tamoxifen-treated patients
FIGURE 2 – DFS (a) and OS (b) curves in the whole cohort of patients stratified according to thymidine phosphorylase (TP) expression.
or CMF-untreated patients (Fig. 4).TP expression had no prognostic value in the group of 68 node-positive patients who did not receive CMF treatment (p ⫽ 0.2942 for DFS; p ⫽ 0.1734 for OS). An association with prolonged DFS but not with OS was observed in the group of patients who did not receive any adjuvant therapy and in the ER-negative cases. DISCUSSION
The present study, in keeping with other biochemical28 and immunohistochemical studies,13,14,19 shows that TP expression is upregulated in the cells of human breast carcinoma. Our present data show a high incidence of TP-positive breast carcinomas, which is higher than the one described by Fox et al.13; this could be ascribed to the different antibodies used in the 2 studies and to the different scoring systems and cut-off points adopted. In our series, high TP was associated with lower tumor grade, positive ER status and lack of p53 overexpression, as well as with prolonged relapse-free and overall
515
THYMIDINE PHOSPHORYLASE IN BREAST CANCER TABLE II – UNIVARIATE ANALYSIS OF DISEASE-FREE SURVIVAL (DFS) BY VARIOUS CLINICOPATHOLOGIC FACTORS1 Factor
Histology Ductal Other Tumor size (cm) ⱕ2 ⬎2 Nodal metastasis Negative Positive Grade 1 and 2 3 ER status Negative Positive p53 Negative Positive TP Negative Positive
No.
DFS No. (%)
p-value
0.3333 149 29
80 (54) 18 (62)
89 85
61 (69) 35 (41)
64 114
45 (70) 61 (53)
89 87
63 (71) 34 (39)
63 112
28 (44) 68 (61)
132 37
76 (58) 15 (41)
39 140
15 (38) 84 (60)
⬍0.0001 ⬍0.0024 ⬍0.0001 0.0163 0.0458
TABLE III – UNIVARIATE ANALYSIS OF OVERALL SURVIVAL (OS) BY VARIOUS CLINICOPATHOLOGIC FACTORS
Histology Ductal Other Tumor size (cm) ⱕ2 ⬎2 Nodal metastasis Negative Positive Grade 1 and 2 3 ER status Negative Positive p53 Negative Positive TP Negative Positive
Subgroup
Adjuvant therapy With CMF2 Without CMF With TAM2 No CMF/no TAM Tumor size (cm) ⱕ2 ⬎2 Nodal status Negative Positive ER status Negative Positive
TP (no.)
p-value
Negative
Positive
For DFS
For OS
11 28 20 10
40 103 62 48
0.0101 0.0523 0.3745 0.0326
0.0096 0.0900 0.1738 0.3414
15 23
74 65
0.0509 0.1324
0.0964 0.0921
11 28
53 86
0.0529 0.0343
0.2937 0.0186
18 20
47 92
0.0355 0.0903
0.0627 0.0754
*: nine patients were treated with CMF and TAM therapy–1TP, thymidine phosphorylase; CMF, cyclophosphamide, methotrexate and 5-fluorouracil; TAM, tamoxifen.
0.0038
1 Statistical analysis was performed using the Kaplan-Meier method, and the p-value was obtained from the log rank (Mantel-Cox) test. ER, estrogen receptor; TP, thymidine phosphorylase.
Factor
TABLE IV – DISEASE-FREE SURVIVAL (DFS) AND OVERALL SURVIVAL (OS) IN DIFFERENT SUBGROUPS WITH UNIVARIATE ANALYSIS1
No.
OS No. (%)
p-value
0.1183 149 29
97 (65) 23 (79)
89 85
70 (79) 48 (56)
64 114
51 (79) 69 (61)
89 87
63 (71) 34 (39)
63 112
32 (50) 86 (76)
132 37
94 (71) 18 (49)
39 140
20 (51) 101 (72)
0.0003 0.0044 ⬍0.0001 0.0007 0.0057 0.0070
1 Statistical analysis was performed using the Kaplan-Meier method, and the p-value was obtained from the log rank (Mantel-Cox) test. ER, estrogen receptor; TP, thymidine phosphorylase.
survival. By studying subgroups of homogeneously staged and treated patients, high TP expression predicts prolonged DFS and OS only in patients treated with adjuvant CMF therapy and, to a lesser extent, in node-positive patients. These 2 latter groups of patients are partially coincident, as the node-positive group of patients included almost all (46/51, 90%) CMF-treated patients. Interestingly, TP had no prognostic value in node-positive patients who did not receive CMF treatment. Our present data, showing that the major prognostic role of TP is restricted to patients receiving adjuvant CMF chemotherapy, are in keeping with the study of Fox et al.,13,20 who showed that TP-positive breast tumors have a significant survival benefit compared with TP-negative tumors when treated with CMF chemo-
therapy. The relationship between high TP and favorable prognosis in patients treated with the CMF regimen is an interesting finding, as it could be hypothesized that high TP could be considered a potential predictor of responsiveness to CMF regimen in breast cancer. This hypothesis has sound biochemical explanations as TP plays a role in the metabolic activation of the pyrimidine antimetabolite 5-FU, which is 1 of the components of the CMF scheme. TP catalyzes the conversion of 5-FU to 5⬘-fluoro-2⬘-deoxyuridine (5⬘-FdUR) by the addition of 2-deoxyribose-1-phosphate,2 and the 5⬘-FdUR metabolites will ultimately inhibit thymidylate synthase.30 The role of TP expression in relation to response to fluorouracil derivatives has indeed been demonstrated in cell lines (colorectal renal cell15,31,32 and pancreatic carcinoma cells33) and in human neoplastic diseases, such as gastric carcinomas.19,34 Moreover, high TP levels may lead to a depletion of the extracellular pool of thymidine, preventing the tumor cells from thymidine rescue by salvage pathway after thymidilate synthase block due to 5-FdUMP and methotrexate.21 These studies suggest that in vivo tumor responsiveness to 5-FU, and possibly methotrexate, is dependent on TP levels, which may suggest a novel approach to therapy: for example, it could be suggested that tumor responsiveness to 5-FU could be modulated by increasing TP expression either by interferon induction31,35,36 or by transfection.37,38 Moreover, TP is also 1 target of prodrug therapeutic strategies aimed at designing drugs that are readily orally absorbed and converted in the active prodrug by enzymes highly expressed in tumors: 1 of these is capecitabine, which is finally converted to 5-FU by TP.39 If the predictive value of TP expression in breast carcinomas will be confirmed by other independent studies, then we could have a promising tool for selection of the most appropriate chemotherapeutic regimen. Further studies are indeed needed, as not all experimental and clinical data fit the above hypothesis,40,41 including a previous study of ours based on in vitro chemosensitivity assay.29 Indeed, TP not only interacts with the 5-FU metabolism but has also a role in the angiogenic process; it could be hypothesized that in different tissues and conditions the prevailing role of TP may be different. In breast cancer, it has been suggested that TP plays a minor role in promoting angiogenesis: TP seems important for remodeling the existing vasculature early in tumor development, consistent with its chemotactic nonmitogenic properties, whereas additional angiogenic factors are needed for completing the angiogenic processes.13 Indeed, in breast carcinomas TP is generally not related to MVD,12,14 as shown also in our present study. It could therefore be hypothesized that TP produced by tumor cells is not crucial for tumor angiogenesis in breast cancer, whereas it plays a major role in pyrimidine metabolism. Conversely, in other tumors such as colorectal carcinoma, TP is
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FIGURE 3 – DFS (a) and OS (b) curves in the group of patients treated with CMF stratified according to TP expression.
more consistently associated with high MVD,42 and its role in pyrimidine metabolism is less important, as suggested by the lack of clinical relation between high TP expression and response to 5-FU therapy in colorectal cancer.41 In conclusion, the present study confirms the study of Fox et al.20 showing that high TP expression is associated with pro-
FIGURE 4 – DFS (a) and OS (b) curves in the group of CMFuntreated patients stratified according to thymidine phosphorylase (TP) expression.
longed survival mainly in patients treated with CMF chemotherapy; as TP has a fundamental role in activating 5-FU, we suggest that TP expression could be an interesting marker for predicting response to 5-FU-based chemotherapy in breast cancer.
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