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© 1999 American Society for Clinical Oncology Immunohistochemical Quantitation of Thymidylate Synthase Expression in Colorectal Cancer Metastases Predicts for Clinical Outcome to Fluorouracil-Based ChemotherapyFrom the Department of Medical Oncology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Department of Pathology, E.O. Ospedali Galliera, Genova, and University of Udine, Udine, Italy; and Wadsworth Center, New York State Department of Health, Albany, NY. Address reprint requests to C. Aschele, MD, Divisione di Oncologia Medica, Azienda Ospedaliera di Padova, Via Giustiniani 2, 35128 Padova, Italy; email c.aschele{at}galliera.it 16132, Genova, Italy; email aschele@hp380.ist.unige.it.
PURPOSE: To determine whether immunohistochemical thymidylate synthase (TS) quantitation predicts for clinical outcome in patients with advanced colorectal cancer treated by fluorouracil (FUra)-based chemotherapy. PATIENTS AND METHODS: TS levels were measured immunohistochemically on archival specimens of colorectal cancer metastases from 48 patients homogenously treated by bolus FUra plus methotrexate alternating with continuous-infusion FUra plus leucovorin. These measurements were retrospectively correlated with patient characteristics and clinical outcome. RESULTS: A significant correlation was found between intratumoral TS expression and all the parameters of clinical outcome analyzed. In patients whose tumors had low (n = 27) and high (n = 21) TS levels, the overall response rates were 67% and 24%, respectively (P = .003). The percentage of tumor shrinkage after chemotherapy was linearly related to TS immunoreactivity (r = .56, P = .00004), and its mean values were 65% and 14% with low and high TS levels, respectively (P = .0001). By logistic regression analysis, low TS expression was the single best predictor of response to chemotherapy (relative probability, 5.0). In patients with low and high TS expression, the median time to progression was 9.6 months v 6.2 months (P = .005) and the median survival time 18.4 months v 15.4 months (P = .02), respectively. Two- and 3-year survival rates were 41% v 15% and 19% v 0% (P = .02), respectively. CONCLUSION: In this cohort of homogenously treated patients, intratumor TS content was a major predictor of clinical outcome. Immunohistochemical TS quantitation provides a convenient, low-cost technique for identifying patients unresponsive to TS inhibitors who may be candidates for alternative chemotherapy regimens.
THYMIDYLATE SYNTHASE (TS) continues to be the main target of colorectal cancer chemotherapy. In particular, the TS inhibitor fluorouracil (FUra) has been the only agent with consistent clinical activity in large-bowel cancer for nearly 40 years.1 Regimens specifically designed to further enhance its effects on TS, with the addition of leucovorin (LV)2 and/or by the development of infusion schedules,3 are the standard treatment for this disease. In addition, a pure TS inhibitor, raltitrexed (Tomudex; Zeneca Pharmaceuticals, Cheshire, United Kingdom),4,5 has been recently approved in many countries for the treatment of metastatic colorectal cancer, and oral fluoropyrimidines4,6-9 are being developed to provide long-term TS inhibition. The ability to identify groups of patients unlikely to respond to TS inhibitors might thus have a substantial clinical impact. Such ability would spare useless toxicity. In fact, the response rate to FUra does not exceed 40% even using LV, infusion schedules, and/or high FUra doses,10-13 and pure TS inhibitors under clinical testing are not more active.14,15 Since new drugs with TS-unrelated mechanisms of action have recently shown activity in this disease, the recognition of patients unresponsive to TS inhibitors might now address even the choice among different treatment options. In particular, the topoisomerase I inhibitor, irinotecan,16-18 and the DNA cross-linking agent, oxaliplatin,19,20 have shown consistent activity in metastatic colorectal cancer and may be used to treat patients unlikely to respond to TS inhibitors. The role of TS in determining fluoropyrimidine cytotoxicity has been clearly established in several preclinical models. High levels of TS activity have been correlated with either acquired or intrinsic resistance to FUra or fluorodeoxyuridine,21,22 and exposure of tumor cells to TS inhibitors has been shown to induce an acute rise in TS levels overcoming drug-induced inhibition.23 A series of clinical observations on small groups of patients provided further support to the general contention that TS expression may critically affect the sensitivity of human tumor cells to fluoropyrimidines.24-26 However, a clear-cut correlation between intratumor TS levels and the clinical response to a single, specific FUra regimen was only recently established.27 Only partial correlations were in fact obtained in the initial clinical studies investigating the predictive role of TS expression, mainly because of the low sensitivity and other technical problems inherent to the biochemical assays that were used (assessment of the catalytic activity or quantitation of the binding capacity for fluorodeoxyuridine monophosphate).25,26 More sensitive methods for quantitating the levels of TS protein and TS gene expression were recently developed.28,29 However, the optimal technique for measuring TS levels in patient tumor samples remains to be defined. Semiquantitative polymerase chain reaction has permitted the measurement of intratumor TS gene expression, which was found to predict for response and resistance to continuous-infusion FUra in a recent study of 42 patients with advanced colorectal cancer.27 Although quite sensitive, this technique requires fresh samples and does not provide a check on the morphology of the sample being tested. The time requirement and costs should also be considered. The development of antibodies to the TS protein has provided the opportunity to quantitate the levels of expression of this enzyme in routinely available, paraffin-embedded tumor samples, using convenient low-cost immunohistochemical assays. Further advantages of this approach are the ability to check for tumor heterogeneity, the lack of contamination of tumor samples with normal tissues, and the suitability for use on archival material. With the use of a monoclonal TS antibody, intratumor TS immunoreactivity was in fact shown to be an independent marker of survival in a retrospective study of patients with radically resected rectal cancer.30 Moreover, TS immunostaining with the same antibody has been shown to correlate with TS mRNA expression in a series of tumor biopsy samples.31 A correlation between TS mRNA and TS protein levels was also found when TS expression was assessed in liver and lung colorectal cancer metastases using a rabbit polyclonal antibody to purified recombinant human TS, which we recently developed.32,33 In the last 5 years, a new clinical regimen alternating bolus with continuous-infusion FUra and using biochemical modulators specific for each schedule of administration was developed at our institute.34 This regimen produced high rates of objective responses in two consecutive phase II studies35,36 and showed a superior activity and efficacy in a randomized comparison with standard-modulated, bolus FUra.37 Still, more than 50% of the patients fail to respond, and attempts to increase its activity have been unsuccessful.36 The present study was thus performed to determine whether immunohistochemical TS quantitation on archival samples of colorectal cancer metastases from patients included in these clinical trials may help identify specific subsets of patients more likely to respond to this regimen.
Patients The study population consisted of 48 patients with metastatic, recurrent, or locally advanced unresectable colorectal cancer, homogenously treated with a regimen of schedule-specific biochemical modulation of FUra (bolus FUra plus methotrexate [MTX] alternating with continuous-infusion FUra plus LV). These patients represent all the patients from two consecutive phase II trials of this regimen35,36 for whom tumor sections from a measurable metastatic lesion were available. The distribution of patient and tumor characteristics for this cohort (n = 48; males/females, 23/25; median age, 61.5 years; age range, 41 to 76 years; median Eastern Cooperative Oncology Group performance status [PS] score, 0) was similar to that of the overall population of the two clinical trials of this chemotherapy regimen (n = 89; males/females, 49/40; median age, 63 years; age range, 41 to 83 years; median Eastern Cooperative Oncology Group PS score, 0) (Table 1).
The treatment consisted of two biweekly cycles of bolus FUra modulated by MTX (given 24 hours earlier) alternating with (14 days later) a 3-week continuous infusion of FUra, modulated by LV (on the first day of each week of infusion). Further treatment details have previously been published.35,36 Response to chemotherapy was assessed according to World Health Organization criteria.38 Briefly, complete response (CR) refers to the disappearance of all disease, partial response (PR) to a greater than 50% reduction in the size of the lesion, and minor response to a 25% to 49% reduction. In addition, for all the patients in these studies, the indicator lesions were measured before the start of chemotherapy and then after each cycle, and a baseline tumor area (expressed in square centimeters as the sum of the products of the maximum perpendicular diameters of all the indicator lesions) was reported along with its variations at each successive cycle. Therapeutic outcome could thus be expressed also as the maximum percentage of reduction in tumor area achieved in each patient. The computed tomography scans of all the patients in these studies were reviewed by an independent radiologist. The rate of objective responses in the current series of patients (CR+PR, 23/48; response rate, 48%; 95% confidence interval, 34% to 63%) was identical to the response rate of the first phase II trial (48%; 95% confidence interval, 31% to 66%35) and only slightly higher compared with the second clinical study (41%; 95% confidence interval, 28% to 55%36). Data on the outcome following chemotherapy with this regimen, along with other details, are reported elsewhere.35,36
Immunohistochemistry Archival, formalin-fixed, paraffin-embedded tissue samples from colorectal cancer metastases were used for this study (liver, 39; lymph nodes, seven; pelvic recurrence, six; peritoneum, four; other, two). Tissue from different metastatic lesions was available from 10 (liver plus lymph nodes, n = 4; liver plus peritoneal implants, n = 3; liver plus pelvic masses, n = 1; pelvic mass plus spleen, n = 1; peritoneal implants plus lymph nodes, n = 1) out of 18 patients with metastases at multiple sites (13 liver plus other sites and five multiple extrahepatic sites). Three patients with pulmonary and hepatic metastases and one patient with hepatic and nodal disease had TS assessed only on liver samples. One patient with liver and hilar node metastases had only the lymph nodes analyzed. TS expression was assessed only on pelvic masses in three patients with concomitant lung or peritoneal disease. Two-micrometer–thick tissue sections were cut, deparaffinized in xylene, rehydrated with graded ethanol, and immersed in Tris-buffered saline (TBS). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in distilled water for 15 minutes. The slides were heated in a microwave oven at 300 W for 10 minutes, cooled, and stored in TBS at pH 7.6. To block nonspecific binding of the primary antibody, a normal rabbit serum (DAKO X901; Dakopatts Co, Glostrup, Denmark) dilution in TBS was used for 20 minutes. After the blocking solution was removed, the TS antibody (2 mg/mL) was applied for 60 minutes in a humidified chamber at room temperature. Negative control studies were performed without applying the primary antibody. The sections were then incubated with biotin-conjugated swine anti-rabbit immunoglobulins for 20 minutes (DAKO-E353), followed by the avidin-biotinylated peroxidase complex for 30 minutes. After the color reaction product was developed with a freshly prepared diaminobenzidine chromogen solution for 5 minutes, the sections were counterstained with light hematoxylin for 1 minute, dehydrated in a series of ethanols, cleared in xylene, and mounted with glass coverslips using Eukitt (O. Kinsler GmbH & Co, Frieburg, Germany). Sections known to stain positively were included in each run as positive controls. Slides were then examined under a light microscope and scored independently by two of the authors (D.D. and S.C.) blinded to both the clinical and pathologic data. Only tumor cells with cytoplasmic staining were counted as positive. TS expression was quantitated using a visual grading system based on the intensity of staining and classified into five groups from 0 (undetectable staining) to 4 (very high intensity of staining). For the purpose of correlation with clinical data, intensity levels 0 to 2 were grouped together and considered low expression (Fig 1A, 1B, and 1C), and levels 3 and 4 were considered high expression (Fig 1D and 1E). Grading TS expression from 0 through 4 did not influence the results described below (data not shown). The agreement in TS evaluation between the two observers was greater than 90%. In the four cases of disagreement, a final score was determined by consensus after re-examination. Seven out of 10 patients for whom paraffin sections from different metastases were available exhibited the same TS status (low v high expression) in all the metastases despite minor differences in the degree of TS immunoreactivity. For the three patients with heterogenous TS levels in different metastases, the highest degree of TS expression was taken to define TS status. Similarly, when heterogeneous levels of TS expression were found within a tumor (nine of 15 cases for which multiple sections from different paraffin-embedded blocks of the same tumor were available), the level of TS expression of that lesion was defined according to the highest TS score that was recorded.
Statistics To assess the relative influence of different prognostic factors on response to chemotherapy, logistic regression analysis with a backward procedure was used. Because of a limited number of events and missing values for some variables, only a limited model could be run. However, this model included all the variables that were predictive of response to chemotherapy by univariate analysis (TS, CEA, PS, and age).
Twenty-one (44%) of 48 patients showed high levels of TS expression, as defined by a TS score equal to 3 and 4 in 16 and five cases, respectively. Among the patients with a low level of TS expression (27 of 48, or 56%), 10 exhibited negative staining for TS, nine had a staining score of 1, and eight had a score of 2. No significant differences in the level of TS expression were found with respect to age, sex, PS, site of the primary tumor, site of metastases, and baseline serum LDH and WBC levels (Table 1). However, TS expression seemed to be related to initial tumor bulk. The median measured baseline tumor area was 18 cm2 and 63 cm2 in patients with low and high levels of TS expression, respectively. Sixty-two percent (15 of 24) of the patients with a measured tumor area greater than the median value for the study (26.94 cm2) had a high TS level, compared with 25% (six of 24) of the patients with an initial tumor area of less than this value (P = .009, Table 1). High levels of TS expression were also marginally associated with increased baseline CEA or AP levels (Table 1; P = .058 and .06, respectively). Accordingly, the median baseline serum CEA and AP levels were 14.40 ng/mL v 47.8 ng/mL (P = .04) and 162 U/L v 252 U/L (P = .025), respectively, in patients with low and high levels of TS expression.
TS expression was inversely correlated with response to chemotherapy (Table 2;
The relationship between TS levels and clinical response remained significant when TS expression was graded from 0 to 4 (response rate: 80%, 66%, 50%, 25%, and 20% with a TS score of 0, 1, 2, 3, and 4, respectively; The median time to achieve an objective response was 2.0 and 3.3 months in patients with low and high levels of TS expression, respectively. Patients with low levels of TS expression also had a longer duration of response (median duration, 7.5 v 4.6 months). The relationship between intratumor TS expression and clinical outcome was not limited to World Health Organization–categorized objective responses. The level of TS immunoreactivity (TS score) was linearly related to the actual tumor shrinkage achieved in each patient (Fig 3; r = .56, P = .00004). Although the actual values of the percentage of tumor reduction achieved in patients with low and high levels of TS expression overlap, the mean values for this figure were 65% and 14% in patients with low and high levels of TS expression, respectively (Fig 4; Student's t test for unpaired samples, P = .0001).
Initial PS score and baseline CEA, LDH and AP levels have been reported to influence response to chemotherapy in advanced colorectal cancer, independently of the agent used.18,35,36,41-43 In this cohort, the initial PS score and the baseline CEA level are the only other variables significantly associated with response to chemotherapy (Table 3). Logistic regression analysis of these prognostic factors indicated that intratumoral TS expression was an independent predictor of response to treatment and the relative probability of achieving an objective response among patients with low TS was 5.0 (Table 4).
Treatment administration and dose delivery seemed to be unrelated to the level of TS expression. The median number of cycles was four versus three, the mean percentage of dose reduction 13% v 24%, and the median dose-intensity of bolus and continuous-infusion FUra was 278 v 270 mg/2/wk and 784 v 848 mg/m2/wk in patients with low and high levels of TS expression, respectively (difference not significant). All patients on this study are now off treatment, 45 of 48 have died, and 44 patients have progressed. One patient is still in CR at 42.8 months. The median follow-up time was 3.6 years, and the median time to progression for all 48 patients was 8.4 months (range, 2 to 42.8 months). Figure 5 shows the influence of the levels of TS expression on the time to progression, with a median time of 6.2 months (range, 2.0 to 15.8 months) in patients high TS levels compared with 9.6 months (range, 3.8 to 43.4 months) in the group with low TS levels (P = .005; log-rank test).
Patients with low levels of TS expression survived longer. The 2- and 3-year survival rates were 41% v 15% and 19% v 0%, and the median survival time was 18.4 months (range, 5.8 to 49.9 months) v 15.4 months (range, 2.0 to 43.1 months) (P = .02; log-rank test) in patients with low and high levels of TS expression, respectively (Fig 6).
Our data demonstrate that immunohistochemical quantitation of TS protein expression in colorectal cancer metastases may help to identify specific subsets of patients with distinctly different outcomes to palliative, FUra-based chemotherapy. The almost 70% response rate observed in the group with low TS levels was two times greater than that observed in the whole cohort of patients treated with this regimen in three consecutive clinical trials (unselected for TS expression).35-37 In addition, TS expression consistently affected all the parameters of clinical outcome under consideration: the higher response rate in patients with low TS levels was paralleled by a significantly greater tumor shrinkage and a longer duration of response and time to progression and translated into a small, but statistically significant, survival benefit. These results compare well to those obtained by other investigators using a polymerase chain reaction–based technique to measure TS mRNA.27 Remarkably, also in that study, the level of response observed in the group with low TS levels was approximately twice that expected in patients unselected for TS expression. While the optimal technique to assess TS in human tumor samples remains to be defined, the potential for widespread use of the immunohistochemical assay that we have used has a major clinical relevance. Other factors have been shown to affect the clinical response to chemotherapy in this disease, including baseline CEA and AP levels, percentage of liver involvement, and initial PS score.18,35,36,41-43 PS was found to predict for response to chemotherapy, almost to the same degree as TS expression, even in the current study. However, the relationship between PS and response to chemotherapy is independent from the agent that is used,18 whereas the predictive role of TS may be unique in that groups of patients could be identified who are unresponsive to a specific class of drugs and may be candidates for alternative forms of treatment. This is of major importance, especially considering the recent development of new drugs with TS-unrelated mechanisms of action that may be used in patients unlikely to respond to TS inhibitors. In fact, a clinical trial is about to be launched of individualized chemotherapy for advanced colorectal cancer based on TS quantitation. The existence of a linear relationship between TS immunoreactivity in tumor samples and tumor response to chemotherapy strengthens the notion that TS status is a major determinant of response to this regimen. On the other hand, TS expression may be a biologic continuum, so that any cutoff may be arbitrary. Whether grouping patients in more than two levels of expression improves the clinical correlations may only be determined in future studies involving a much larger number of patients. In this study, high levels of TS expression were observed more frequently in patients with increased baseline serum CEA and AP levels. To our knowledge this issue has not been addressed in previous studies and needs further investigation. Because CEA and AP levels may reflect the actual bulk of disease42-43 and because TS expression in this study was also associated with the measured baseline tumor area, one may argue that the relationship between TS expression and clinical outcome merely reflects the greater responsiveness of patients with a lower tumor burden and good clinical conditions. However, by logistic regression analysis, the predictive ability of TS expression for response to chemotherapy was independent from other important prognostic factors, including PS score and CEA levels. In addition, there were no differences in compliance with treatment or dose delivery between patients with low and high TS levels that might account for the different response rates in these two groups. Baseline pretreatment TS levels are not the only determinant of TS inhibition. The rate of fluorodeoxyuridine monophosphate formation,44 as well as the rise in the competing pool of deoxyuridylate,45 the availability of 5,10-methylenetetrahydrofolate in the more effective polyglutamylated form,46,47 and the activity of the FUra catabolizing enzyme dihydropyrimidine dehydrogenase48 have been shown to influence both the degree and the duration of TS inhibition. In addition, an acute induction of TS levels has been observed after treatment with TS inhibitors, leading to a recovery of enzyme activity.23 Molecular factors modulating the pathways of cellular death downstream of TS inhibition,49-51 as well as clinical variables generally affecting response to chemotherapy in advanced colorectal cancer,41-43 may also mitigate the predictive ability of TS expression for the clinical outcome. Considering all these factors, it is not surprising that neither TS immunoreactivity nor TS mRNA levels27 could fully predict for response to chemotherapy. In the present study, the predictive value of a low level of TS expression for a good clinical outcome seemed to be superior to that of high levels of expression for a poor outcome. Although no progressions were reported in the first group, five patients with high levels of expression obtained a partial response. In contrast, in the trial measuring TS mRNA levels, a threshold value could be identified above which no responses were obtained.27 The relatively weaker association between high TS levels and resistance to chemotherapy in our study may reflect the hybrid nature of the FUra regimen that we have used.34 The antitumor activity of MTX-modulated bolus FUra has been shown to depend on the incorporation of FUra metabolites into RNA, and it may be unaffected by TS levels.34,45 Patients with high levels of TS expression may thus still be responsive to the bolus segment of our regimen. On the other hand, the prediction of low levels of TS expression for a good clinical outcome was stronger in the current study, as two thirds of the patients with low TS immunoreactivity achieved an objective response, all but three patients achieved at least a 25% reduction in tumor mass, and no patient progressed on therapy. In contrast, an approximately 50% failure rate has been reported among patients with low TS mRNA levels.27 This discrepancy may reflect the potential ability of immunohistochemical assays to identify high levels of TS expression in patients who seem to express low TS levels with other techniques. Although a rough correlation between TS protein and gene levels has been shown,31,33 translational regulation may have a substantial role in determining the levels of expression of this enzyme.52 Immunohistochemistry may thus give a more direct and accurate estimate of TS protein content compared with mRNA quantitation. Immunohistochemical assays also allow consideration of the histologic features of the sample analyzed, enabling detection of its contamination with normal tissues, which may be an important confounding factor. Finally, immunohistochemical assays permit assessment of TS expression in archival specimens from different metastatic lesions. This may be of critical importance in patients with multimetastatic colorectal cancer. In another study from our group, a substantial intrapatient heterogeneity in TS levels was indeed observed between primary colorectal tumors and the corresponding distant metastases and even among different metastatic lesions (data not shown). Of interest, this heterogeneity might explain the negative findings obtained in a recent study of patients with advanced colorectal cancer. In this study, TS levels measured immunohistochemically on archival samples of previously resected primary tumors failed to predict the response to chemotherapy for advanced disease.53 This study is thus the first to demonstrate that the levels of TS protein expression, measured with a convenient, low-cost immunohistochemical technique on currently available archival specimens of colorectal cancer metastases, predict the clinical response to a specific FUra regimen. Although retrospective, these results are particularly promising considering that all the patients were homogenously treated in two consecutive phase II trials, with an independent review of the responses and a measurement of the maximum percentage of tumor shrinkage achieved in each patient. Based on multivariate analysis, our data suggest that TS immunoreactivity is the single best predictor of response and may thus be used to select patients unresponsive to this regimen for treatment with alternative drugs. Given the consistent results obtained in two independent studies that used completely different techniques, TS expression should also be considered before randomization in future clinical trials comparing different forms of treatment for advanced colorectal cancer. Further studies to confirm these results in a larger cohort and to test whether the schedule of FUra administration may influence the relationship between TS expression and response to treatment are required. In addition, since patients unlikely to respond to TS inhibitors may now be offered alternative forms of treatment, studies investigating the effects of TS levels on response to non–TS-targeted drugs are also warranted.
Supported by grants from the Ministero della Sanità 1997, Associazione Italiana per la Ricerca sul Cancro, National Research Council of Italy 1998, National Research Council of Italy Biotechnology, and National Cancer Institute/Department of Health and Human Services (grant no. CA44355).
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 or Fisher's exact test, as indicated. The association between TS expression and the proportion of responses was assessed using the Mantel test for trend. The baseline area of the measured tumor lesions and its maximum percentage of reduction following treatment, as well as the baseline serum levels of carcinoembryonic antigen (CEA), lactate dehydrogenase (LDH), alkaline phosphatase (AP), and WBCs, were compared between patients with low and high levels of TS expression using the Student's t test for unpaired samples. Survival and progression-free survival were calculated from the onset of chemotherapy until death or progression, respectively. Patients without events were censored at the date of the last follow-up. One patient who discontinued chemotherapy and underwent resection of his liver metastases, with two reoperations upon recurrence, was censored at the time of surgery. For overall survival, death from any cause was considered as an event. Progression-free and overall survival curves were constructed using the Kaplan-Meier method, and differences were assessed by the log-rank test. 
5FU vs schedule specific biochemical modulation in advanced colorectal cancer. Proc Eighth Int Congress Anti-Cancer Treat 1998, p 96 (abstr O98) 
