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Clinical Trials |
Tumour and Angiogenesis Research Group, Department of Radiotherapy and Oncology, Medical School, University of Thessalia, Larissa 41222, Greece [M. I. K.]; Department of Pathology, Democritus University of Thrace, Alexandroupolis 68100, Greece [A. G., E. S.]; Department of Medical Oncology, Hellenic Co-operative Oncology Group, AHEPA Hospital, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece [G. F.]; Department of Cellular Science and Imperial Cancer Research Fund Medical Oncology Unit, Oxford Radcliffe Hospital, Headington, Oxford OX3 7LJ, United Kingdom [K. C. G., A. L. H.]
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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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The importance of TP and TS in cancer chemotherapy with antimetabolites has focused our attention recently. In vitro data suggest that high levels of TP correlate with an enhanced response to fluoropyrimidines, because increased 5-fluorouracil transformation to fluorodeoxyuridine results in higher amounts of FdUMP production, increased DNA FdUTP incorporation, and strong TS inactivation by ternary complex formation. TP may also prevent thymidine salvage, and therefore, TP can potentiate 5-fluorouracil or methotrexate from a different mechanism besides activation (4) . On the other hand, high levels of TS correlate with decreased sensitivity to fluoropyrimidines. If high levels of TS remain free after fluoropyrimidine chemotherapy, DNA synthesis and cancer cell proliferation will efficiently go on. Although several clinicopathological studies have been reported recently, providing evidence for an important role of TS in resistance to antimetabolites (5 , 6) , the role of TP has not been studied extensively (7) .
TP is identical to platelet-derived endothelial cell growth factor, which is a protein involved in the stimulation of endothelial cell migration and proliferation (8, 9, 10) . The association of TP expression with increased intratumoral angiogenesis has been verified in several studies in breast (11) , lung (12) , and colorectal cancer (13) . Experimental data support the idea of an important synergistic role of antiangiogenic agents with radiotherapy (14) . Indeed, in a recent study, we observed an important role of angiogenesis in the radiotherapy and chemotherapy outcome of head and neck carcinomas (15) . Moreover, we noted a direct association of TP and mutant p53 expression with high angiogenesis in squamous cell carcinomas (16) . It may, therefore, be suggested that although TP is a target for antimetabolite chemotherapy, its angiogenic properties are potentially of importance in defining the outcome of cytotoxic and radiation therapy. Nevertheless, our recent observation that TP overexpression in less vascularized non-small cell lung cancer cases correlates with a worse prognosis, suggesting that TP may also confer an aggressive tumor behavior through pathways not necessarily linked to the intratumoral vascularization density (17) . The patterns of TP immunohistochemical cancer cell reactivity may be nuclear and/or cytoplasmic (16, 17, 18) , and the differential pathogenetic or prognostic role of these subcellular patterns of expression has never been studied in the past.
It seems, therefore, that the role of TP in defining prognosis and response to cytotoxic and radiation therapy has several mechanisms. In the present study, we examined the subcellular patterns of TP expression in locally advanced squamous cell head and neck cancer and their correlation with the intratumoral angiogenesis. The role of TP expression in defining survival and response to platinum/5-fluorouracil chemotherapy and radiotherapy was also investigated. All previous studies have analyzed pretreatment variables, but it is well recognized that marked changes in oxygenation and proliferation can occur after radiotherapy. We therefore measured changes of TP expression and localization in a subset of patients undergoing radiotherapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. All cases were locally advanced
cancers and were treated with conventionally fractionated radiotherapy
combined with chemotherapy. None of the patients underwent surgery. The
follow-up of patients ranged from 1 to 108 months (median, 12 months).
For patients alive the follow-up ranges from 6 to 108 months (median,
48 months).
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Treatment and Response Assessment.
Baseline studies included physical examination, chest X-rays, whole
blood count with differential and platelet count, complete biochemical
profile, bone scan, and computed tomography of the cranium, neck, and
chest. Treatment characteristics are shown in Table 2
. Although the four treatment cohorts
included in this study were not randomized, these were sequential in
time, and there was no patient selection according to the regimen.
Thirty-eight patients received three cycles of induction chemotherapy,
followed by radical radiotherapy (LINAC 6 MV or Cobalt 60 Unit; 2
Gy/fraction; five fractions/week; total tumor dose, 70 Gy).
Fifty-six patients were treated with concurrent chemoradiotherapy (same
radiotherapy regimen).
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Assessment of TP Expression.
TP expression was assessed with the P-GF.44C monoclonal antibody
(18)
using the APAAP method. Two-µm paraffin-embedded
sections were dewaxed and rehydrated. P-GF.44C monoclonal antibody as
undiluted supernatant was applied at room temperature for 30 min and
washed in TBS. Rabbit antimouse antibody 1:50 was applied for 30 min,
followed by application of mouse APAAP complex 1:1 for 30 min. After
washing in TBS, the last two steps were repeated for 10 min each. The
color was developed by 15-min incubation with New Fuchsin solution.
Normal rabbit IgG was substituted for primary antibody as the negative
control (same concentration as the test antibody). Tumor-infiltrating
macrophages were used as a positive internal control.
In a previous study, we observed that TP staining in squamous lung and
head and neck cancer could be either cytoplasmic or nuclear (16
, 17)
. Tissue samples were scanned at low (x40 and x100) power,
and the percentage of nuclear (TPnuc) and strong
cytoplasmic (TPcyt) expression was separately assessed in
six chosen optical x250 fields of the highest reactivity. The
percentage of staining was the mean value of the scores obtained. We
also assessed the overall TP staining (TPall), which is the
percentage of positive cells no matter whether the reactivity was in
the nucleus or in the cytoplasm or in both subcellular localizations.
The results were analyzed as continuous variables. We also considered
the mean percentage of positive cells as a cutoff point to group cases
in two categories of low and high TPnuc or TPcyt
reactivity. Fig. 1a
shows a
case with predominantly nuclear reactivity; Fig. 1b
shows a
case with both nuclear and cytoplasmic reactivity.
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The specimens were scanned at low optical power (x40 and x100), and microvessel counting was performed on x250 fields. Three areas (per case) of high vascularization were chosen for microvessel counting. The overall MS was the mean value of all appraised fields of the case. Vessels with a clearly defined lumen or well-defined linear vessel shape but not single endothelial cells were taken into account for microvessel counting. Overall MS was used for statistical analysis, both as a continuous and a categorical variable. Microvessel analysis was performed by three independent observers, and results were assessed for interobserver variability. Disagreement was resolved from a conference microscope. The vascular grade grouping of our cases was based on a previous study (15) . Briefly, patients with an intermediate microvessel score (10–50 vessels per x250 field) had a statistically significant better CR rate, local relapse, and OS as compared with patients with poor (<10 vessels per x250 field) or high (>50 vessels per x250 field) vascularization. Cases with poor and high vascularization had a similarly poor prognosis. Therefore, in the present study, we adopted three groups of vascular grade: LVG, MVG, and HVG.
Statistical Analysis.
Statistical analysis and graphs were performed using the GraphPad Prism
2.01 and the Instat 3.0 packages (San Diego, CA). Nonparametric
unpaired two tailed t test with Welch’s correction or
Fisher’s exact t test was used for testing relationships
between categorical tumor variables, as appropriate. Linear regression
analysis was used to assess interobserver variability or correlation
between continuous variables. Multiple regression analysis was also
used as appropriate. Survival curves were plotted using the method of
Kaplan and Meier, and the log-rank test was used to determine
statistical differences between life tables (20
, 21)
. The
end points for analysis were the response rate, the local
progression-free survival, and the OS starting from the last day of
radiotherapy. CR rate was separately assessed from PR rate because head
and neck cancer is a curable disease, with the PR always indicative of
treatment failure. A Cox proportional hazard model was used to assess
which of the tumor variables were independently correlated with
response, local relapse, and death events (22)
. All
Ps are two sided, and P <0.05 was used for
significance.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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None of the TP reactivity patterns showed a statistical significant correlation with T stage, N stage, histological grade, or localization of the primary tumor (data not shown).
TP Reactivity and MS.
The overall MS ranged from 6 to 83 microvessels per x250 optical field
(mean, 34.2 ± 21; 95% CI, 29–38). Interobserver variability was
minimal (P < 0.0001, r >0.91). Linear
regression analysis of TP score and MS showed a significant association
of TPnuc with increasing MS (P < 0.0001,
r = 0.45). TPcyt was marginally associated
with MS, and a poor goodness of fit was noted (P =
0.01, r = 0.24). Multiple regression analysis of
TPnuc, TPall, and TPcyt showed that only
TPnuc was significantly associated with the MS
(P = 0.02; t ratio, 2.35).
TP Reactivity and Response.
In a bivariate model, we analyzed the association of TPnuc
and TPcyt reactivity with response to radiotherapy and
chemotherapy (all cases). Only the nuclear patterns of staining showed
a significant association with response (P = 0.04;
t ratio, 2.0).
We analyzed the percentage of TPnuc-reactive cells in tumors
with CR, PR, or less than PR (NR) to radiotherapy and chemotherapy.
Cases with CR after combined therapy (all cases considered together)
had a significantly lower percentage of TPnuc reactivity
(27.1 ± 23) as compared with cases with PR (42.0 ± 30;
P = 0.03) and NR (35.9 ± 25; P =
0.17; Fig. 2a
).
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).
Similarly, a lower percentage of TPnuc cells was present in
cases that had a CR after concurrent chemoradiotherapy
(P = 0.05). Analysis of cases that showed PR/NR to
induction chemotherapy but CR after radiotherapy that followed the
induction chemotherapy showed no significant difference in the
percentage of TPnuc reactivity as compared with cases that
had PR or NR after radiotherapy. The CR rate of this latter category of
patients was lower as compared with the CR rate observed after
concurrent chemoradiotherapy [12 of 30 (40%) versus 33 of
56 (59%); P = 0.11].
Table 3
shows the responses (CR, PR, and
NR) obtained after radiotherapy, induction chemotherapy, radiotherapy
after induction chemotherapy, and concurrent chemoradiotherapy
stratified for TP nuclear reactivity.
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Fig. 3
shows the Kaplan-Meier survival
curves (all cases) for LRFS and OS, stratifying for TPnuc
reactivity and using as a cutoff point of the mean percentage of cells
with nuclear staining (32%). The median LRFS for patients with high
TPnuc reactivity was 5 months as compared with 32 months of
median LRFS obtained in patients with low TPnuc reactivity
(P = 0.001). The median OS in patients with high and
low TPnuc reactivity was 10 and 36 months, respectively
(P = 0.0005).
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Nuclear TP Reactivity, MS, and Prognosis.
T stage, N stage, histological grade, or primary site localization was
not associated with response or prognosis both in terms of LRFS and OS.
A higher CR rate, a better LRFS, and OS have been observed in cases
with MVG (MS 11–50) as compared with cases with LVG (MS <11) and HVG
(MS >50). This finding has been reported and discussed in a previous
study (17)
. In a bivariate model taking into account the
VG (MVG versus LVG, HVG) and TPnuc reactivity
(low versus high), we observed that, despite the previously
noted association of TPnuc with high angiogenesis,
TPnuc reactivity was an independent factor defining response
to radiotherapy (P = 0.01; t ratio, 2.5),
whereas VG was not (P = 0.22; t ratio,
1.20). As far as the local relapse events is concerned, both
TPnuc and VG were independent prognostic variables
(P = 0.0007, t ratio 3.5 for
TPnuc; P = 0.0001, t ratio 5.0
for VG). Similarly, both parameters had an independent prognostic
meaning as for death events (P = 0.003, t
ratio 2.99 for TPnuc; P = 0.0001,
t ratio 4.0 for VG).
Comparative Analysis of MS and TPnuc Changing
Patterns before and after Radiotherapy.
Sections from head and neck carcinomas treated with 20 Gy of
radiotherapy showed bands and islands of cancer cells immersed within
avascular degenerated tissue areas that were frequently of high
vascularization and of intense nuclear TP reactivity (Fig. 4
). Degenerated cancer cells
were negative for nuclear TP expression, whereas cytoplasmic expression
was occasionally present. In patients with CR, the mean MS in these
areas of viable cells was 35.3 ± 19 before radiotherapy and
decreased to 19.3 ± 15 after radiotherapy (P =
0.11). In patients with PR/NR, the initial score was 39.5 ± 23
and was maintained at 42.5 ± 32 (P = 0.81).
Similarly, the mean TPnuc reactivity in patients with
CR was decreased from 40.2 ± 32 before radiotherapy to 28.1 ± 27 after radiotherapy (P = 0.05). In patients with
PR/NR, this remained stable (50.1 ± 1 versus 50.1 ± 34; P = 1).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ominous prognostic value of high intratumoral vascularization has been reported by a large number of studies in different tumors (23) . Because TP is associated with high vascularization, its overexpression should be associated with worse prognosis. However, the role of TP in the outcome of chemotherapy and radiotherapy is balanced by at least three different TP-related features: (a) antimetabolite activation; (b) increased intrinsic cancer cell aggressiveness; and (c) increased tumor vascular density and angiogenicity.
In the present study, we examined the response to platinum and 5-fluorouracil induction chemotherapy in a cohort of 38 squamous cell head and neck cancers. Cases that showed a CR after induction chemotherapy had a very low percentage of TP nuclear and cytoplasmic reactivity as compared with cases with PR or NR. It seems, therefore, that cancer cells with very low levels of TP are sensitive to DNA-damaging agents. Impaired DNA repair may underlie the phenomenon. The observation that induction chemotherapy with 5-fluorouracil did not result in a higher response rate in TP-overexpressing cases may be a consequence of the relative importance of platinum activity in TP-deficient cells. In a recent study by Fujieda et al. (24) , susceptibility to 5-fluorouracil cytotoxicity in oropharyngeal cancer was observed only in extremely high TP expression. Our study shows that TP expression is not a major factor defining favorable response to 5-fluorouracil.
The predictive role of nuclear TP reactivity was also observed in a cohort of 56 patients treated with concurrent chemoradiotherapy and overall in all patients treated with radiotherapy. LRFS and OS were significantly affected by TP overexpression. The median LRFS was 42 months in cases with reduced TP expression versus 5 months in cases with TP overexpression. Similar results have been reported by Metzger et al. (25) , where increased TP levels in colorectal cancer were associated with poorer response to chemotherapy. Because radiotherapy and both platinum and 5-fluorouracil exert their cytotoxic activity through DNA damage, either cross-linking or single and double strand breaks, it may be that DNA-damaging agents are more effective in TP-deficient cancers. Although Fujieda et al. (24) did not find TP levels associated with resistance to cisplatin, in our study we used combined modality therapy with radiation and also 5-fluorouracil with cisplatin.
A possible alternative explanation for why tumors with high TP had a poorer response to therapy may be suggested from the comparative study of angiogenesis and TP expression, in tumors before and after 20 Gy of radiotherapy. The most frequent patterns of tissue structure in irradiated tumors was the presence of viable cancer cell foci immersed within degenerate tumor areas. The MS and the percentage of TP-positive cells in viable tumor areas were significantly decreasing after radiotherapy in tumors that reached CR, whereas these were stable or even increased in cases that did not respond completely to radiotherapy. This observation may show that the ability of cancer cells to maintain active angiogenesis and TP pathways may be important for the results of radiotherapy. The concept of "rapid cancer cell repopulation" during radiotherapy has been suggested to explain the reduced control rate after overall treatment time prolongation (26, 27, 28) . However, the nature of the phenomenon is unclear because the cancer cell potential doubling time and the cell cycle time are unlikely to decrease during radiotherapy (29, 30, 31) . Whether the so called "rapid tumor repopulation" phenomenon is a result of a more complicated process involving angiogenic pathways is an issue raised that should be investigated further.
Recently, novel antiangiogenic therapies have emerged (14 , 32) . Several studies suggest that antiangiogenesis approaches may significantly improve the efficacy of radiotherapy. After administration of angiostatin concurrently with radiotherapy a substantial increase of tumor control is achieved, although no cancer cell radiosensitization is mediated (14) . In a recent study by Gorski et al. (33) , up-regulation of vascular endothelial growth factor was shown to accrue after cancer cell irradiation, and the use of anti-vascular endothelial growth factor antibodies, together with radiotherapy, significantly decreased the growth rate of experimental tumors. Specific inhibitors of TP have also been produced and tested experimentally (34) . Such compounds may be of importance in abrogating the radioresistance of tumors with high TP expression, whether this is related to intrinsic cell factors or to the angiogenic process.
We conclude that TP is an important molecule associated with angiogenesis, with resistance to radiotherapy and chemotherapy, and with poorer survival of locally advanced squamous cell head and neck cancer treated with chemoradiotherapy. Although TP is one pathway for metabolic activation of 5-fluorouracil, it seems that TP-related angiogenicity and a possible sensitivity of TP-deficient tumors to DNA-damaging agents may also confer two distinct mechanisms that should be further studied to clarify the role of the protein in tumor behavior. The cancer cell ability to maintain high angiogenesis and TP expression during radiotherapy may also prove of predictive value. The present study provides a strong rational for subsequent clinical trials of concurrent use of radiotherapy and chemotherapy with antiangiogenic agents and with specific TP inhibitors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Designed, analyzed, interpreted, and financially
supported by the Tumor and Angiogenesis Research Group and the Imperial
Cancer Research Fund. 
2 To whom requests for reprints should be
addressed, at Tumour and Angiogenesis Research Group, 18 Dimokratias
Avenue, Iraklion 71306, Crete, Greece. Phone: 30-932-480808; Fax:
30-81-392848; E-mail: targ{at}her.forthnet.gr
3 The abbreviations used are: TP, thymidine
phosphorylase; TS, thymidylate synthase; MS, microvessel score; CR,
complete response; PR, partial response; NR, no response; APAAP,
alkaline phosphatase/anti-alkaline phosphatase; MAb, monoclonal
antibody; LVG, low vessel grade; MVG, medium vessel grade; HVG, high
vessel grade; CI, confidence interval; LRFS, local relapse-free
survival; OS, overall survival.
Received 9/ 7/99; revised 11/16/99; accepted 11/22/99.
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REFERENCES |
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