1 Departments of Medical Oncology and Pathology, EO Ospedali Galliera, Genoa; 2 Azienda Ospedaliera di Parma, Parma; 3 Ospedale San Salvatore, Pesaro; 4 Azienda Ospedaliera TreviglioCaravaggio, Treviglio; 5 Ospedale S Maria delle Croci, Ravenna; 6 Ospedale Infermi, Rimini; 7 Azienda Ospedaliera di Padova, Padua, Italy; 8 Wadsworth Center, New York State Department of Health, Albany, NY, USA
Received 14 January 2002; revised 30 April 2002; accepted 17 May 2002
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Abstract |
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Different 5-fluorouracil (5-FU) schedules and/or biochemical modulators may result in different mechanisms of cytotoxicity, potentially affecting the correlation between thymidylate synthase (TS) expression and the clinical response to the fluoropyrimidine.
Patients and methods:
TS levels were measured immunohistochemically on archival specimens of colorectal cancer metastases from 124 patients homogeneously treated in a series of clinical trials at our institutions with: (A) leucovorin (LV)-modulated infusional 5-FU (n = 48); (B) LV-modulated bolus 5-FU (n = 41); (C) methotrexate (MTX)-modulated bolus 5-FU (n = 35).
Results:
A statistically significant correlation between TS levels and the clinical response was observed with the regimens involving continuous infusion and/or LV modulation (response rate in patients with low and high TS: 66% versus 24%, P = 0.003, and 50% versus 0%, P = 0.0001, in group A and B, respectively). Conversely, TS levels failed to predict the clinical response within the group of patients treated with MTX-modulated bolus 5-FU (response rate 21% versus 13%, P = 0.50, with low and high TS, respectively). Consistently, the median time to progression/overall survival time in patients with low and high TS were 9 versus 6 months/19 versus 14 months (P = 0.009/0.035, group A), 8 versus 2 months/12 versus 6 months (P = 0.002/0.0006, group B) and 3 versus 2 months/12 versus 13 months (P = 0.14/0.74, group C).
Conclusions:
The correlation between intratumoral TS levels and the clinical response to 5-FU depends strongly on the schedule of administration/biochemical modulators that are used in different 5-FU regimens. These data strengthen the notion that different 5-FU schedules have different mechanisms of cytotoxicity.
Key words: biochemical modulators, colorectal cancer, 5-fluorouracil, response prediction, schedule of administration, thymidylate synthase
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Introduction |
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The role of TS expression as a determinant of fluoropyrimidine cytotoxicity has been established in both preclinical and clinical models. In vitro, high levels of TS activity have been correlated with either acquired or intrinsic resistance to 5-FU or fluorodeoxyuridine [12, 13] and exposure of tumor cells to TS inhibitors has been shown to induce an acute rise in TS levels which may overcome drug-induced inhibition [14]. On clinical grounds, patients with low levels of TS gene or protein expression in their colorectal cancer metastases have shown response rates (RRs) to fluoropyrimidine-based chemotherapy that are 310 times higher compared with those obtained in patients with high TS levels in multiple recent studies [1519].
Considering the complex activating pathways and multiple intracellular targets of the fluoropyrimidine [20], this clear-cut correlation is somehow surprising. An improvement in overall response prediction was indeed obtained with the simultaneous assessment of TS and different enzymes involved in 5-FU metabolism [21, 22]. As to the target, we have previously shown that TS inhibition is the dominant site of action with prolonged exposures to the fluoropyrimidine, while incorporation into RNA plays a major role with pulse dosing [23]. Biochemical modulators may also shift the mechanism of action of 5-FU towards a specific cellular target: LV selectively potentiates TS inhibition while methotrexate (MTX) has been shown to enhance 5-FU incorporation into RNA [2426].
Response prediction based on the assessment of intratumoral TS expression may thus be limited to infusional schedules, in particular with LV modulation, while the clinical activity of bolus regimens may be independent from the level of expression of this enzyme. To test this hypothesis, we have used an immunohistochemical technique suitable for archival material to measure the level of TS protein expression in colorectal cancer metastases from a series of patients homogeneously treated with one of three different 5-FU regimens (bolus or infusion, LV or MTX modulation). These measurements were then retrospectively correlated with patients characteristics and a series of parameters of clinical outcome [RR, time to progression (TTP), overall survival (OS)] both on data pooled from all chemotherapy regimens and separately within each treatment group.
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Patients and methods |
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Patients treated with regimen A received a 3-week continuous infusion of 5-FU modulated by weekly LV alternated to bi-weekly administrations of bolus 5-FU modulated by MTX (given 24 h earlier). Regimen B consisted of daily administrations of bolus 5-FU modulated by LV repeated for five consecutive days every 4 weeks. The third regimen (C) consisted of bi-weekly courses of bolus 5-FU modulated by MTX (given 24 h earlier). Further treatment details have been previously published [2730]. The cases included in this study represent all the patients from a series of clinical trials of these regimens for whom tumor sections from a measurable metastatic lesion were available. The rates of objective responses according World Health Organization criteria [31], observed in the three groups of patients analyzed in this study (48%, 17% and 17% in group A, B and C, respectively) are similar to those originally reported for the entire patient populations treated with the corresponding 5-FU regimens (48%, 41% and 33% with the infusional regimen [2729], 10% with LV-modulated bolus 5-FU [30] and 14% with MTX-modulated bolus 5-FU [29]).
The main patient and tumor characteristics of each treatment group are reported in Table 1. One-hundred and five (85%) patients had a primary tumor located in the colon and 19 (15%) in the rectum. Fifty-nine patients had metastatic disease limited to the liver, 22 had liver metastases plus other sites of disease and 43 had extra-hepatic disease. At baseline, the median area of the measured tumor lesions was 25 cm2 (range 21234 cm2) and the median white blood cell count (WBCC) was 7515 cells/mm3 (range 420014 900 cells/mm3). The median baseline serum levels of carcinoembryonic antigen (CEA), lactic dehydrogenase (LDH) and alkaline phosphatase (ALP) were 17.2 ng/ml (range 0.717 400 ng/ml), 312 U/l (range 363652 U/l) and 219 U/l (range 601614 U/l), respectively. Fifteen of 124 patients had previously received adjuvant treatment following resection of the primary tumor (14 chemotherapy, one pelvic radiation therapy).
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Statistics
Analyses were conducted on data pooled from all chemotherapy regimens and separately within each treatment group. The association between TS expression and dichotomized clinical variables, including response to treatment, was analyzed using the chi-square or Fishers exact test as indicated by the data. The baseline serum levels of CEA, LDH, ALP and WBCC were compared between patients with low and high levels of TS expression using the Students t-test for unpaired samples. Progression-free and OS curves were constructed using the KaplanMeier method and differences were assessed by the log-rank test.
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Results |
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The mean and median levels of immunoreactivity (TS score) were similar among the three patient groups as shown in Table 2. Consistently, the proportion of patients with high levels of TS expression was 44%, 66% and 46% in groups A, B and C, respectively.
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Discussion |
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The present study confirms the predictive value of TS expression in a large series of 124 patients but clearly indicates that the results are limited to specific 5-FU regimens. Patients with low TS levels had a significantly higher probability of response to infusional and/or LV-modulated 5-FU (regimens A and B) with longer TTP and OS times compared with patients with high TS. In contrast, intratumoral TS levels failed to predict clinical outcome in a group of patients treated with MTX-modulated bolus 5-FU (regimen C). Although the RR to this regimen was slightly higher in patients with low compared with those with high TS, this difference was not statistically significant, with substantial overlapping of the CIs, and was substantially smaller compared with that observed with the other regimens. Furthermore, TS level segregated almost absolutely both complete responders and progressors following treatment with regimens A and B while for regimen C treatment failures were equally distributed in the groups with low and high TS and the only CR was observed in the latter cohort. Consistently, for patients treated with MTX-modulated bolus 5-FU, TTP and OS following chemotherapy were found to be independent of the level of TS expression. Finally, the mean level of TS expression was similar between responders and non-responders in the group of patients treated by MTX-modulated bolus 5-FU while TS immunoreactivity was significantly lower in responders compared with non-responders treated with the other two regimens.
Since this was a retrospective study, the distribution of patients among the three treatment cohorts was not controlled and patients and tumor characteristics may not have been evenly balanced between the three treatment groups. However, all of the patients had been previously accrued in a series of prospective clinical trials with similar eligibility requirements including the need for measurable disease. Indeed, the main patient and tumor characteristics were fairly balanced among the three groups except for a higher proportion of patients with intra-abdominal recurrences in group B and a higher percentage of patients who had received previous adjuvant chemotherapy in group C. In particular, the median age ranged from 60 to 64 years, the median PS from 0 to 1 and the proportion of patients with liver-limited disease from 34% to 58%. In addition, group C patients had the same median PS, the same relative proportion of patients with a primary rectal tumor and a similar proportion of patients with liver metastases as compared with patients treated with regimen A. It is therefore unlikely that the different dependence on TS expression observed for the activity of the three regimens investigated in this study depends on the distribution of patient characteristics in the three treatment groups.
These data indicate that TS inhibition does not represent a major target for MTX-modulated bolus 5-FU. This is consistent with preclinical data showing that the mechanism of action of 5-FU depends on the schedule of administration. We have previously demonstrated that TS inhibition is the main mechanism of action with prolonged exposures to low doses of the fluoropyrimidine, while incorporation into RNA becomes the main site of action with pulse-dosing [23, 33, 34]. In addition, while LV specifically potentiates the TS inhibitory activity of 5-FU by stabilizing the lethal ternary complex that consists of fluorodeoxyuridylate, 5,10-methylenetetrahydrofolate and the enzyme itself [24], pretreatment with MTX has been shown to enhance the incorporation of the fluoropyrimidine into RNA by increasing phosphoribosylpyrophosphate pools and, therefore, the activation of 5-FU to fluorodeoxyuridine triphosphate (FUTP) [25, 26]. A recent clinical observation showing a significant decrease in TS and ß-actin mRNA following the administration of bolus 5-FU lends support to this contention [35]. Our findings contrast with the results of a recent study that showed a correlation between intratumoral TS levels and the clinical response to a regimen of MTX-modulated bolus 5-FU [36]. However, in that study TS expression was assessed on primary colorectal cancer rather than metastatic samples. This may be a major difference as we have previously shown that TS levels in primary tumors are neither related to TS expression in the corresponding metastases nor predictive of their response to palliative chemotherapy [37]. In addition, in the study by Paradiso et al. [36], patients with negative TS immunostaining were compared with patients with any grade of positive immunoreactivity while in the present study patients with negative and low immunoreactivity were more conventionally grouped and compared with patients with high levels of immunostaining. Finally, even in that study the predictive value of TS expression for the clinical response to MTX-modulated 5-FU was small with a borderline statistical significance and a lack of correlation with TTP and OS. This latter point implies a weak potential to predict tumor response as it has been demonstrated that objective responses are associated with increased survival in this disease [38].
Surprisingly, LV-modulated bolus 5-FU proved to be the regimen where the difference in RR between patients with low and high TS was more marked. In particular, the prediction of high TS levels for resistance was absolute in that all the patients with high TS failed to achieve an objective response. Based on previous preclinical data showing that bolus 5-FU acts mainly via incorporation into RNA [23], TS levels were not anticipated to predict the clinical response to this regimen. This unexpected pattern of prediction may have multiple explanations. LV modulation may shift the mechanism of action of bolus 5-FU from incorporation into RNA to TS inhibition. Stabilization of the ternary complex may in fact result in prolonged retention of inhibition even after a short-term exposure to the drug [39]. The significant increase in the level of clinical activity with a doubling of the rates of objective tumor responses obtained with the addition of LV to bolus 5-FU along with the changes observed in the spectrum of toxicity [34] lend support to the notion that LV-modulated and unmodulated bolus 5-FU may have distinct biochemical targets. However, based on the present data it is impossible to determine whether or not TS levels also affect the clinical activity of unmodulated bolus 5-FU and this aspect may be difficult to investigate even in future studies. The use of bolus 5-FU without biochemical modulators has in fact been progressively abandoned in the last 10 years and metastatic tumor samples from patients treated with this regimen may be difficult to retrieve. An additional explanation for the strong TS dependence of this regimen is that bolus 5-FU is administered for five consecutive days with a possible further increase in the duration of TS inhibition.
Based on preclinical data, the highest degree of correlation was expected for infusional schedules. Conversely, only a three-fold difference in RR was observed in the present study between patients with low and high TS treated with regimen A, with 20% of the patients with high TS levels actually achieving an objective response. This may reflect the hybrid nature of the infusional regimen used in this study. LV-modulated infusional 5-FU was in fact alternated with MTX-modulated bolus 5-FU [28]. As previously discussed, the antitumor activity of MTX-modulated bolus 5-FU has been shown to depend on the incorporation of 5-FU metabolites into RNA and it may be unaffected by TS levels [34]. The 20% RR observed in patients with high TS levels may thus reflect the responsiveness to the bolus part of this hybrid regimen. An absolute correlation between high TS levels and resistance, with a threshold value for TS expression above which no responses were obtained, was in fact observed in a previous study investigating a pure LV-modulated infusional 5-FU regimen [15]. Consistent with this hypothesis, the proportion of patients with high TS achieving an objective response in the present study was similar in the group of patients treated with the hybrid regimen and in the group who received MTX-modulated bolus 5-FU.
These data have important clinical implications. The specificity for infusional and/or LV-modulated 5-FU regimens emphasizes the predictive value of intratumoral TS expression for response to the fluoropyrimidine. If the correlation between TS levels and the clinical response merely reflected the more aggressive biological behavior of tumors with high TS, the same correlation would have been observed with every 5-FU regimen and even with other drugs. In line with our results, TS levels in colorectal cancer metastases failed to predict the clinical response to CPT-11, confirming the high drug-specificity of this correlation [40]. Based on the present data, TS quantification cannot be used to predict the clinical response to MTX-modulated bolus 5-FU. Despite a decreased popularity in advanced disease, this regimen is alive and well and a randomized comparison with the Machover regimen involving over 2000 patients has been recently completed in the adjuvant setting [41]. In addition, trimetrexate (TMQ) may soon replace MTX as a modulator of bolus 5-FU resulting in increased activity and lower toxicity [42]. Putative markers of response to this regimen include the level of expression of FUTP-catabolizing enzymes and the assessment of early RNA dysfunction following 5-FU administration [35, 43]. Finally, the present findings support the use of MTX-modulated bolus 5-FU in patients with TS-related resistance to the fluoropyrimidine. In particular, patients failing infusional 5-FU may be treated with combination regimens including MTX- or TMQ-modulated bolus 5-FU. New combinations incorporating CPT-11 or oxaliplatin into these regimens should therefore be developed.
In conclusion, this study demonstrates that the prediction of clinical outcome based on the assessment of intratumor TS expression is limited to specific 5-FU regimens. This study is also the first to confirm on clinical grounds that TS is not the main target for MTX-modulated bolus 5-FU, strengthening the notion that the mechanism of action of the fluoropyrimidine depends on the schedule of administration and the nature of the biochemical modulators. This may be of major clinical importance to optimize the development of combination regimens that integrate 5-FU and new drugs like CPT-11 and oxaliplatin. The importance of the schedule of 5-FU administration in determining the toxicity of these combination regimens is indeed already emerging [44].
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Acknowledgements |
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Footnotes |
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