To the best of our knowledge, this is the first study to show a correlation between FPGS and DHFR expression and 5-FU sensitivity results in NSCLC. FPGS showed a stronger correlation with 5-FU sensitivity than DHFR.
FPGS converts intracellular folic acid and folic acid antagonists, such as methotrexate, into polyglutamic acid, which is retained intracellularly for a long time. Polyglutamylation of intracellular 5,10-methylenetetrahydrofolate enables more efficient formation and stabilization of inhibitory ternary complexes involving TS and metabolites of 5-FU, and it may also increase the cytotoxicity of 5-FU.15 Two reports have shown a correlation between 5-FU sensitivity and FPGS in colon cancer15 and breast cancer.16
DHFR is the target enzyme of methotrexate, which enters the cell, tightly binds to DHFR, and inhibits the reduction of dihydrofolate to tetrahydrofolate.17 Only one report has evaluated the activity of DHFR and the antitumor effect of 5-FU.18 They reported that DHFR mRNA had a high value using a 5-FU-resistant mouse cell line.
5-FU is activated only after it is converted to 5-fluorodeoxyuridine monophosphate (FdUMP). TS, an enzyme that is essential for DNA synthesis, methylates deoxyuridine monophosphate (dUMP) and converts it to deoxythymidine monophosphate (dTMP). Therefore, FdUMP is covalently bonded to TS together with 5,10-methylenetetrahydrofolate (5,10-CH2-THF), which is reduced to folic acid, to form a strong ternary complex consisting of dTMP and deoxythymidine triphosphate (dTTP), which inhibits DNA synthesis.19 FPGS acts on the pathway that converts 5,10-CH2-THF from monoglutamate to polyglutamates, the increase in which is indispensable for creating a ternary complex that may affect the sensitizing effect of 5-FU. The function of TS activity is inhibition, and the pool of dTTP, which is a precursor of DNA, is depleted, leading to inhibition of DNA synthesis and cell death.19
In this study, the positive correlation between TS expression and 5-FU sensitivity is the opposite of the results of other reports of NSCLC.5, 20-22 Regarding the sensitivity of TS in gastric cancer and colorectal cancer, sporadic reports have shown no correlation or reverse correlation between the sensitivity to 5-FU and increased TS activity.23, 24 In NSCLC, a positive correlation with TS was reported, but TS activity tends to be lower than in other carcinomas,14 suggesting that even if TS activity is high, it falls within the range where the effect of 5-FU can be observed.
Regarding the sensitivity of 5-FU, the activities of OPRT and DPD have been well evaluated, and several reports show that they are involved in the sensitivity of NSCLC.25-27 The results of this study did not show a correlation between OPRT and DPD expression and 5-FU sensitivity. Although the OPRT/DPD ratio has been reported to be an important predictor of the efficacy of fluoropyrimidine-based chemotherapy for metastatic colorectal cancer,25 this report on DPD/OPRT showed no significant difference with the sensitivity of 5-FU.
Several reports have described the relationship between TS, DPD, and OPRT and 5-FU sensitivity in NSCLC. Eguchi et al.20 evaluated the relationship between response to treatment and immunohistochemical expression levels in patients with advanced NSCLC. Low expression levels of DPD and TS were seen in patients not treated with S-1-carboplatin, which is associated with better response and longer survival in patients treated with paclitaxel-carboplatin. Tumor expression levels of TS and DPD predict the response to S-1-carboplatin chemotherapy in patients with advanced NSCLC. Nakano et al.21 reported an immunohistochemical study on the clinical importance of TS, OPRT, and DPD expression using 151 NSCLC specimens resected from patients treated postoperatively with tegafur/uracil (UFT). Patients who had tumors with low TS expression (p = 0.0133), high OPRT expression (p = 0.0145), or low DPD expression (p = 0.0004) had significantly high of 5-year survival rates. Shintani et al.5 investigated patients using RT-PCR for intratumoral expression and examined the correlation between gene expression and the efficacy of 5-FU in NSCLC. Patients receiving postoperative 5-FU alone (n = 30) comprised the 5-FU group, and those who had only surgery were included in the control group (n = 86). When dichotomized by mean TS and DPD mRNA levels, patients with low DPD tumors receiving 5-FU had significantly better prognosis than those who did not receive adjuvant treatment (p = 0.041). Based on these results, quantification of TS and DPD mRNA levels can predict the efficacy of 5-FU after surgery in patients with NSCLC.
5-FU is rapidly degraded by DPD. Due to the higher DPD activity in NSCLC compared to other carcinomas,14 5-FU alone is less effective, necessitating co-administration of CDHP, for which S-1 was developed. In view of the mechanism of action of 5-FU, the effects of 5-FU are expected to be reduced if expression of the target enzyme TS and the degrading enzyme DPD in tumor tissue is high. CDHP (Gimeracil), which is used in S-1,28 inhibits DPD. In this study, we found no difference in the sensitivity results even if CDHP was added to 5-FU in the preliminary sensitivity test. This suggests that DPD may not affect the antitumor effect of 5-FU in vitro.
There are several reports that The HDRA method correlates well with the susceptibility of NSCLC to anticancer drugs and its clinical efficacy. 29-31 Further, the usefulness of HDRA has been documented for several other cancer types including gastric cancer32 and colorectal cancer.33 This histo-culture method has the advantage of culturing cancer cells while maintaining cell-cell contacts which has good cell viability, and the disadvantage of requiring a certain amount of tissue sample. In the present study, sufficient amount of sample could be obtained from the surgical specimens. Moreover, the high evaluability rate (n=419, 96.5%) from previous tests for NSCLC conducted at our institution demonstrate that this method is a good alternative for testing the sensitivity of 5-FU in NSCLC.
The limitations of this study are the small number of cases, the single-institution design, and the in vitro results of the anticancer drug sensitivity test. Further clinical studies are needed. In the future, studying the relationship between anticancer effects in NSCLC patients who actually received 5-FU and the expression levels of various factors in the tumors is necessary. Another limitation is the in vitro use of specimens obtained during surgery. For unresectable advanced NSCLC, small specimens such as those obtained from bronchoscopy should be used. The feasibility of such transbronchial lung biopsy samples is being investigated. Nakajima et al.34 used a metastatic lymph node sample obtained with endo-bronchial ultrasound-guided transbronchial needle aspiration in patients with non-small cells to obtain TS, DPD, TP, and OPRT mRNA. The feasibility of expression analysis should be evaluated. Clinical application is also expected.
Few reports have examined the sensitivity of 5-FU in NSCLC. Our study provides results that will be useful for assessing the sensitivity of 5-FU in future clinical applications.