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Letters in Drug Design & Discovery, 2004, 1, 45-49

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Thymidine Phosphorylase Over-Expression in Oral Squamous Carcinoma Tissue as a Potential Target of Capecitabine G. Ranieri*, R. Patruno, G. Fiore1, G. Saponaro, A. Paradiso and L. Grammatica National Cancer Institute of Bari, Bari, Italy 1Department

of Internal Medicine, Immunology and Infectious Disease, Section of Internal Medicine, University of Bari Medical School, Bari, Italy Received October 5, 2003: Accepted October 29, 2003

Abstract: Advanced oral squamous carcinoma (OSC) is typically treated with 5Fluorouracil (5-FU) based regimens. Capecitabine (CAP) is a thymidine phosphorylase (TP) activated oral fluoropyrimidine, rationally designed to generate 5-FU preferentially within tumours. The high activity of CAP in intestinal and breast cancer suggests that CAP may have a role in the therapy of OSC. This tumour selectively is achieved through exploitation of the significantly higher activity of TP in tumour compared with healthy tissue. In the present study, the epithelial and macrophages TP expression were significantly higher in OSC than in non-dysplastic oral leukoplakia (NDOLP) (p=0.004, p=0.005; respectively). Because OSC is sensitive to 5-FU, and TP expression is significantly higher in OSC than in NDOLP, TP-activated CAP could be a promising therapy worthy of clinical investigation.

Keywords: thymidine phosphorylase, capecitabine, 5-Fluorouracile, oral squamous carcinoma, leukoplakia, immunohistochemistry. INTRODUCTION Thymidine phosphorylase (TP), also known as platelet derived endothelial cell growth factor (PD-ECGF), is an enzyme involved in thymidine synthesis and degradation, catalyzing the breakdown of thymidine to thymine and the sugar 2-deoxy-D-ribose 1-phosphate, which exerts a clear angiogenic action by stimulating endothelial cell migration [1,2]. TP was first described almost 45 years ago and purified in the mid-1970s both from Escherichia coli and Salmonella. Escherichia coli TP is a homodimer of 45 kDa subunits [3-6]. Eukaryotic TP, a 47 kDa subunit homodimer, was first purified to homogeneity from the human amniochorion [7,8]. In humans, immunohistochemical localization of TP has been demonstrated in epithelial cells, endothelial cells, macrophages and mast cells [9]. In vitro data suggest that high levels of TP correlate with an enhanced response to fluoropyrimidines, because increased 5-fluorouracil transformation to fluordeoxyuridine results in higher amounts of fluoro-2’-deoxyuridine-5’-monophosphate production and increased DNA fluoro-2’-deoxyuridine-5’triphosphate incorporation. A putative role for TP in human malignancies has been suggested since the early 60s [10]. N4–pentyloxycarbolnyl–5’-deoxy-5-fluorocytidine, commonly called capecitabine (CAP), a TP-activated fluoropyrimidine was rationally designed to generate 5-FU preferentially at the tumour site [11]. After oral administration, CAP crosses the gastrointestinal barrier and is completely absorbed. It is subsequently converted into 5FU in a three-step mechanism involving several enzymes. In *Address correspondence to this author at the National Cancer Institute, 70126 Bari-Italy, Via Amendola 209; Tel: +39 080 5555331; Fax: +39 080 5555 677; E-mail: [email protected] 1570-1808/04 $45.00+.00

the first step, it is metabolized into 5’-deoxy-5fluorocytidine (5’-dFCR) by hepatic carboxylesterase. 5’dFCR is then deaminated into 5’-deoxy-5-fluorouridine (5’d5-Furd) by citidine deaminase, mainly localized in liver and tumour tissue. Finally 5’d5-Furd is transformed into 5FU under the action of TP in tumour tissue [11]. This tumour selectivity is achieved through exploitation of the significantly higher activity of TP in tumour tissue compared with healthy tissue. Because CAP mimics continuous infusion of 5-FU and has demonstrated considerable activity in breast and colon cancer, it has attracted interest as treatment for other tumour types known to be sensitive to 5-FU or in which TP is up-regulated [12,13]. In this way CAP should have two main advantages: first it should increase the concentration of the active principle at the tumour site; second, it should decrease the concentration of drug in healthy tissues with a consequent reduction in systemic toxicity. The present study analyzed epithelial and stromal TP expression in OSC and NDOLP tissue as a potential target of CAP. MATERIALS AND METHODS Patients From a consecutive series of patients treated surgically at the Otorhinolaryngoiatry Unit of Istituto Oncologico of Bari, formalin-fixed paraffin-embedded samples of oral mucosa from 21 patients with NDOLP and 50 patients with OSC (T1-3 N0-1 M0) were selected [14,15]. Clinical NDOLP was histopathologically characterized by hyperparakeratosis and papillary hyperplasia, but no dysplasia was observed. All the paraffin-embedded blocks selected underwent preliminary evaluation to confirm the presence of © 2004 Bentham Science Publishers Ltd.

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Ranieri et al.

pathologically representative tissue. OSC p a t i e n t characteristics are shown in Table 1. The definitive histological diagnosis was made on haematoxylin and eosinstained slides. Table 1.

Clinicopathological Characteristics of 50 Patients with OSC Variable

No. of patients

≥ 62 years

26

0.90 (K) for the variable evaluated. All calculations were performed using the SPSS package (SPSS, Inc., Chicago, Illinois).

Tumour category pT1

7

pT2

23

pT3

20

pN0

26

pN1

24

Nodal status

Cytohistological grade

RESULTS

G1

13

G2

29

G3

8

TP expression was evaluated in all 50 samples of OSC (Table 1) and all 21 samples of NDOLP.

Immunohistochemical Assay Six µm-thick serial sections of formalin-fixed, paraffinembedded OSC and NDOLP tissue were deparaffinized by the xylene-ethanol sequence. For antigen retrieval the sections were microwaved at 500W for 10 minutes, after which endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution. Slides were incubated with monoclonal antibody anti-TP (P-GF.44C Neo-Markers, Fremont, California) diluted 1:100 for 1h at room temperature. The bound antibody was visualized using biotinylated secondary antibody, avidin-biotin peroxidase complex, and 3-amino-9-ethylcarbazole or 3,3diaminobenzidine. Nuclear counterstaining was performed Table 2.

TP immunoreactivity was observed in normal epithelial cells “Fig. (1)”, malignant epithelial cells “Figs. (2-4)” , macrophages “Fig. (2)” and endothelial cells. The usual pattern of positive staining was both cytoplasmic and nuclear “Figs. (3, 4)” but only one of these was also observed “Figs. (1, 2)”. TP immunoreactivity was heterogeneous and rarely focal within a section. The mean percentage of positive epithelial cells for TP expression (TPexpressing cells) was significantly higher in OSC “Figs. (24)” than in NDOLP “Fig. (1)” (53±26% and 18±17%, respectively; p=0.004). In the same manner the mean percentage of positive TP macrophages was significantly higher in OSC than in NDOLP (16± 9 and 10± 8 respectively; p=0.005; Table 2). In the series of OSC samples, the median value of 60% positive malignant cells and 20 positive macrophages cells were utilized to

* Mean ± Standard Deviation. Macrophages and Epithelial TP Expression as a Function of Histopathological Diagnosis Macrophages TP (Number of positive cells at 400x fields)

Epithelial TP (Percentage of positive cells at 400x fields)

Oral Squamous Carcinoma n=50

*16 ± 9

*53% ± 26%

Non-Dysplastic Oral Leukoplakia n=21

*10 ± 8

*18% ± 17%

p value (t-test)

p=0.005

0.004

Thymidine Phosphorylase Over-Expression

Fig. (1). NDOLP: low epithelial TP expression. Arrow indicates a single epithelial cell with cytoplasmic TP immunoreactivity (red colour) and no nuclear TP expression (x 400; Chromogen 3-amino-9-ethylcarbazole).

distinguish between high versus low TP expression in tumour tissue. The association both of epithelial and macrophages TP expression and important clinical pathological characteristics was analyzed. The percentage of tumours with high TP expression was similar in younger and older patients, in both sexes, among pT1, pT2 and pT3 tumours, in positive and negative nodes and in tumours with different cytopathological grade. With regard to clinical outcome, we analyzed relapse and death at a follow up of 5 years at a median time of 18 months. No differences in time to progression (TTP) and Overall Survival (OS) curves were found for patients with high versus low TP expression (p=n.s.).


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Fig. (3). OSC: high TP expression. Single arrow indicates a strong nuclear TP expression (brown colour). A moderate epithelial cytoplasmic TP expression is also present. Double arrow indicates an interstitial area as a negative internal control: no TP immunostaining is observed (x400; Chromogen: 3,3diaminobenzidine).

Fig. (4). OSC: high TP expression. Single arrow indicates a strong TP expression in a bi-lobed nucleus (brown colour; x400; Chromogen: 3,3-diaminobenzidine).

Fig. (2). OSC: single arrow indicates a stromal macrophage with TP immuno-reactivity (red colour); double arrow indicates a group of tumoural cells with TP expression. Many scattered lymphocytes with blue nucleus negative for TP expression (x 400; Chromogen: 3-amino-9-ethylcarbazole).

DISCUSSION TP has a multifunctional role; as an enzyme it is involved in nucleotide salvage and is associated with 5fluorouracil activation [1, 20]. It is also a pro-angiogenic

factor by 2-Deoxy-D-ribose and increased TP expression hasbeen correlated with increased angiogenesis [16, 18]. The evidence that TP may play a role as a chemotactic endothelial agent supports our previously published data that this enzyme participates in the restructuring and optimization of the OSC vascular bed [9,21]. TP overexpression has been reported in pre-malignant lesions such as cervical intraepithelial neoplasia, and in several solid tumours such as breast, colorectal, bladder, stomach, and lung [22-26]. In the present study, a significant increase in immunoreactive TP epithelial and macrophage cells was found in OSC with respect to NDOLP. This suggests that TP expression increases in concomitance with the tumourigenesis of oral mucosa. Several prognostic studies are published on TP expression in head and neck cancer, however, very little data are available on OSC as compared to NDOLP [27-29]. In a study on 58 patients with oral and oropharyngeal squamous cell carcinoma, OS was worse in the group with high compared to low TP-expressing

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tumours (p= 0.03), however carcinoma cells with high TP were sensitive to 5-FU in spite of poor prognosis [30]. Koukourakis reported that, in 94 patients with locally advanced squamous cell head and neck cancer, only the group with low TP-expressing tumours was associated with better OS (p=0.0005) and sensitivity to cytotoxic and radiation therapy [10]. Our study did not correlate TP expression with prognostic significance or with clinical factors such as sex, age, cytohistological grade, tumour size or nodal status. These different results could be explained by the TP immunoreactivity pattern and cell type, we evaluated. Both tumour cells and macrophages can, in fact, be positive for TP expression and can participate to generate intratumour TP activated CAP. Consequently, in our analysis both cell type were considered. Furthermore, the T P immunoreactivity pattern observed was heterogeneous: mixed nuclear and cytoplasmic, only cytoplasmic or only nuclear, and all these cells were evaluated in our work. In Fujieda's study only TP positive tumoural cells with mixed pattern was considered in the evaluation. Conversely, Koukourakis performed a separate analysis of the percentage of nuclear and cytoplasmic cells in TP expression but also evaluated the percentage of mixed nuclear and cytoplasmic TP-expressing cells. In this study, only low nuclear TPexpression correlated with a better OS. So the standardization of the method to detect TP expression is a very important point to further research; again no analysis of NDOLP was performed in the above studies. For what concerns the crucial role of TP in cytotoxic drugs activation, experimental data indicated that in vitro carcinoma cells with high TP expression were sensitive to 5FU [10]. Because CAP mimics continuous–infusion 5-FU and has demonstrated considerable activity in breast and colon cancer, it has attracted our interest as potential treatment of OSC [22, 31-33]. In fact OSC is sensitive to 5FU and TP over expression in OSC suggest a potential utilization of CAP in this tumour type. In addition the expression levels of TP in OSC tumour tissue may represent a predictive factor to select the patients more sensitive to CAP. The crucial role of TP in the activation of CAP provides a strong pre-clinical rationale for evaluating CAP for the treatment of OSC [34]. Because OSC is sensitive to 5-FU, and TP expression is significantly higher in OSC than in NDOLP, TP-activated CAP could be a promising therapy worthy of clinical investigation.

Ranieri et al.

T1-3

=

Tumour Size (TNM Classification)

N0-1

=

Lymph-Nodal Status (TNM Classification)

M0

=

Metastasis Status (TNM Classification)

pT1

=

Pathological Tumour Size (pTNM Classification)

pT2

=


pT3

=


5’-dFCR

=

5’-deoxy-5-fluorocytidine

5’d5-Furd

=

5’-deoxy-5-fluorouridine

500W

=

500 Watt

s.d.

=

Standard Deviation

p

=

Statistical Significance

n.s.

=

No Significance

TTP

=

Time To Progression

OS

=

Overall Survival

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

ABBREVIATIONS

[16]

OSC

=

Oral Squamous Carcinoma

5-FU

=

5-Fluorouracile

CAP

=

Capecitabine

TP

=

Thymidine Phosphorylase

PD-ECGF

=

Platelet Derived Endothelial Cell Growth Factor

[19]

NDOLP

=

Non-Dysplastic Oral Leukoplakia

[20]

Kda

=

Kilo-Dalton

DNA

=

Deoxyribo-Nucleic Acid

[17] [18]

[21]

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