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Many drugs can be used for adjuvant therapy of breast cancer, including anthracyclines, cyclophosphamide, 5-fluorouracil (5-fU) and, recently, taxanes (TXT) have shown promising results. 5-FU blocks thymidylate synthase (TS) which cross-links p53 mRNA, inhibiting its synthesis. TS overexpression is one of the main mechanisms involved in 5-FU drug resistance. Enough p53 mutations can confer resistance to chemotherapy using anthracyclines and 5-FU, while are associated with improved responses to TXT. The aim of this study was to examine the TS and p53 levels in tumor samples and to compare the efficacy of FEC (5-FU, epirubicin, cyclophosphamide) and TXT chemotherapy in a group of patients with differing TS and p53 status. We examined 84 breast tumor samples using immunohistochemistry. TS and p53 levels were inversely related, and TS and p53 positivity was significantly associated with the failure of FEC treatment and with a good response to TXT therapy (p

10546 Background: Adjuvant chemotherapy is used in the treatment of breast carcinoma independently of axillar node involvement. Different drug combinations such as CMF, FAC, FEC are still used; recently new drugs such as TXT (NEJM 332:1004,1997) show activity and are used also in adjuvant chemotherapy. 5 Fluorouracil (5Fu), a drug involved in main therapeutic regimens, blocks Thymidylate Synthase (TS), an enzyme involved in the DNA synthesis. TS not only links its own mRNA, but also p53 mRNA, inhibiting post transcriptional p53 protein synthesis. TS protein overexpression (Cancer Res 55:1407,1995), and/or its absence (Cancer Res 61:1421,2001) are some of the main mechanisms of 5-Fu drug resistance; moreover TS protein could inhibit p53 protein synthesis. Many clinical studies demonstrate that tumours with mutations of p53 are resistant to anthracyclines chemotherapy, 5Fu and radiotherapy (Carcinogenesis 17:1417,1996). Other studies show that the relationship between p53 and taxanes chemosensitivity seems to be more complex, because p53 mutations are associated with better taxanes drug response (Nature Med 2:255,1996). Aim of the study is to find association between TS and p53 levels, and relation with CMF or TXT treatment. Methods: We detected TS and p53 protein levels with immunohistochemistry assay in 84 paraffin embedded tumour samples from untreated patients (pts) who underwent surgery for primary breast cancer. Stage was T2N2M0 and pts were divided into two groups depending on the drug treatment received. One group (44 pts) was treated by 6 cycles of adjuvant CMF chemotherapy, the other by 4 cycles TXT alone. Results: 30% of pts showed lower TS and higher p53 levels, and had better TXT, while worse CMF treatment response; 30% of pts showed higher TS and lower p53 levels and all had worse response to all treatments; 40% of pts showed intermediate TS and p53 levels and different response to both CMF or TXT. Conclusions: On the basis of these results we identified a sub-group of pts showing lower TS and higher p53 levels that could be treated by a first line therapeutic regimen including Taxanes, not by drug combinations including 5 FU. No significant financial relationships to disclose.

In view of the structural similarity of mitoxantrone to anthracyclines and its ability to intercalate into DNA, we studied its influence on the synthetic processes of DNA and proteins in CD-1 mice tissues. By studying at the DNA level the impairment of 2H-thymidine incorporation and its return to normal, it was found that bone marrow and spleen showed similar behavior, i.e., a rapid return to normal, which occurred before bone marrow cell number and spleen weight returned to basal values. At the cardiac level, the incorporation values of precursors into DNA, reduced by treatment with mitoxantrone, came back very slowly to the control ones. Hepatic DNA showed a lower sensitivity to mitoxantrone. Analysis of 3H-leucine incorporation into three protein fractions of the heart showed that the contractile proteins were the most responsive fractions to mitoxantrone treatment. Experiments on CD-1 mice treated repeatedly with mitoxantrone revealed that the antitumor drug, at the cumulative dose of 8 mg/kg i.v., induced alterations in myocardiac morphology similar qualitatively to those induced by doxorubicin, although smaller quantitatively.

Breast cancer is a heterogeneous disease, so therapeutic predictive biological markers need to be identified. To date an accurate evaluation of predictive markers is mainly done at the primary site; however, the main goal of adjuvant therapy for breast cancer is the control of micrometastases. The aim of this study is to assess as therapeutic and/or prognostic marker, the proliferation status of primary tumors and involved nodes as measured by Ki67 and thymidylate synthase (TS) expression, in 30 breast cancer node positive patients. TS is the main target of 5-fluorouracil (5-FU) activity, and its overexpression is one of the mechanisms of 5-FU drug resistance; however, in some studies its absence is responsible for a worse response to 5-FU. Our results show that malignant cells of involved nodes were in a post mitotic phase of the cell cycle, and show a low proliferation index and TS expression, while the primary tumours and controls, were strongly positive. On these basis we can hypothesize that these cells could be less sensitive to 5-FU. Further studies are necessary to identify other mechanisms responsible for their metastasing capability and/or for their aggressiveness.

We tested the novel NF-kappaB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) in the hepatic cancer (HCC) HepG2, HA22T/VGH and HuH-6 cells. The sensitivity to the cell growth inhibitory and apoptotic effects of the agent increased along with the levels of constitutively activated NF-kappaB, which were low in HepG2 and higher in HA22T/VGH and HuH-6. In HA22T/VGH, DHMEQ exhibited synergy with cisplatin. In the same cells, DHMEQ exerted dose-dependent decreases in the nuclear levels of activated NF-kappaB and attenuated NF-kappaB activation by cisplatin. It down-regulated Bcl-XL mRNA in a dose-dependent manner and up-regulated that of Bcl-XS. It also decreased interleukin 6 (IL-6), NAIP and, after 16 h of exposure to the higher concentration tested (10 microg/ml), c-IAP-1 mRNA levels. At 10 microg/ml it caused significant increase in Bax, XIAP, cyclin D1 and beta-catenin mRNAs. The combination of DHMEQ with cisplatin produced unexpected significant decrease in c-IAP-2 and Bcl-XS mRNAs as well as additive decrease (IL-6, NAIP and, after 16 h, Bcl-XL) or increase (XIAP at 8 h) in gene expression. HA22T/VGH produce IL-6; in agreement with the results on mRNA, DHMEQ inhibited such a process. HA22T/VGH lack the IL-6 receptor alpha chain, ruling out that in these cells the antitumor effects of DHMEQ may be attributed to an interference with a growth stimulatory autocrine loop based on IL-6. However, the use of DHMEQ in HCC might be beneficial to contrast the adverse systemic effects of the released cytokine.

5-Fluorouracil (5FU) is the main drug used for the treatment of colorectal cancer (CRC) and Thymidilate Synthase (TS) is its target enzyme. TS gene has regulatory tandemly repeated sequences in its 5'' and 3''untraslated region (5''-3'' UTR). CRC often shows a kind of genomic instability called Microsatellite Instability (MSI) that is associated with TS levels and survival. Our data show that the genotype 2R/2R (homozygosity for 2 tandem repeat sequences in the 5''UTR) is more frequently associated with MSI+ and lower TS levels. More over we did not find any significant association between the 2R/3R (heterozygosity for 2 and 3 tandem repeat sequences in the 5''UTR) and 3R/3R (homozygosity for 3 tandem repeat sequences in the 5'' UTR) genotypes with the MSI+ and MSI-, while these genotypes were associated with a higher TS expression. As a consequence we can hypothesise that patients bearing CRC with the MSI+, the 2R/2R genotype and with low TS levels could have a better prognosis and they could not be drug resistant.

Microsatellite instability (MSI) is a characteristic feature of up to 15% of colorectal cancers (CRC) and is associated with better response to adjuvant chemotherapy with 5-fluorouracil (5-FU). In this study we have investigated the association between the MSI status and the mRNA expression as well as the polymorphisms of the cellular target of 5-FU therapy, thymidylate synthase. Polymorphisms in the 3'- and the 5'-UTR of the TS gene were determined by a PCR assay in 53 colorectal cancer tissues. TS mRNA was quantified by real-time RT-PCR. Data were correlated with the MSI phenotype. There was neither a significant correlation between the polymorphisms in the TS gene and the MSI phenotype nor between the mRNA expression and MSI status. CRC with a 3R/3R or 2R/3R genotype showed a significantly higher TS mRNA expression than those with 2R/2R genotype (p=0.001 and p=0.026, respectively). No association was seen between the polymorphism of the 3'-UTR and mRNA expression. From our results, we conclude that there is no association between MSI status and TS expression. Samples containing the 3R/3R or 2R/3R genotype of TS seem to have higher mRNA levels perhaps due to a higher mRNA stability. Polymorphic variants of the 3'-UTR do not influence the TS mRNA level. Genotyping of the 5'-UTR and quantitation of TS mRNA levels might serve as predictors for the response to 5-FU based chemotherapy.

Thymidylate Synthase (TS) is the key enzyme for DNA synthesis pathways and is inhibited by 5-fluorouracil (5FU). The aim of this work was to study TS expression and the proliferation rate in the different histological types of colorectal carcinoma (CRC). 50 patients with CRC were included in this study and evaluated immunohistochemically using the monoclonal antibodies, TS106 and Ki67. 20 tumours were of the intestinal type, 15 cases were signet ring cell carcinoma (SRCCs) and 15 cases were "mixed-type", with at least two different histological components. Intestinal and mucinous histotypes were positive for TS and Ki67, while "signet ring cell" samples were negative or showed only weak and focal positivity for both the TS and Ki67 antibodies. Our results show that signet ring cells (that are also often present in intestinal and mucinous carcinomas), are in the post-mitotic phase of the cell cycle and show a low proliferation index and TS expression. As TS is the key enzyme for DNA synthesis pathways and is inhibited by 5-fluorouracil (5FU), we can hypothesise that TS expression levels in the different histotypes of CRC could affect the potential responsiveness of these tumours to fluoropyrimidine chemotherapy, with a low efficacy being expected in signet ring cell areas.

To study GP-170 in superficial bladder cancer at initial diagnosis and at recurrence and to evaluate if intravesical chemoprophylaxis modifies the expression of GP-170 in tumor recurrences. GP-170 was retrospectively assessed in 160 patients affected by primary superficial transitional cell carcinoma of the bladder and followed for up to 10 years. Eighty-four patients (52.5%) recurred after transurethral resection (TUR). Adjuvant intravesical chemotherapy after TUR was adopted in 52 patients. The correlations between GP-170 and G-grade, T-category, risk of recurrence and of progression, and adoption of adjuvant intravesical chemotherapy were investigated. The correlations between variations in grade and stage at recurrence and modifications in GP-170 expression were also studied. No significant correlation between GP-170 expression and G-grade and T-category was found. A significant correlation was detected between GP-170 expression and recurrence (P=0.0383). It showed a biphasic pattern, i.e., tumors that did not express GP-170 had a higher recurrence rate, but high GP-170 levels were also associated with an increasing risk of recurrence. Intravesical chemotherapy did not induce significative variations in GP-170 expression. No correlation was found between progression and GP-170. GP-170 seems to be an independent prognostic factor for recurrence in superficial bladder tumors. A negative GP-170 pattern and high levels of GP-170 are associated with an increasing risk of recurrence but have no impact upon progression. In our experience, GP-170 is neither induced nor modified by intravesical chemotherapy, although it might represent a factor of chemoresistance when strongly expressed.

Structural changes in the macromolecular targets of pharmacological agents can result in alterations in the efficacy of these agents. In previous studies Berger et al. (1) identified a variant structural form of thymidylate synthase (TS) that is associated with relative resistance to 5-fluoro-2'-deoxyuridine, in a human colonic tumor cell line. They observed that expression of the variant TS, which differs from the normal form by a tyrosine to histidine substitution at residue 33, confers a 4-fold level of drug resistance in mammalian cells, as well as in bacteria. Now we report on the use of RT-PCR techniques to see if that variant TS form could be present in human samples from patients who underwent surgery for primary colorectal cancer and been previously untreated and to try to find relationships between that hypothetical variant TS form and the 5-Fluorouracil treatment. The possible role of Tyr-33 in 5-fluoropyrimidine-mediated inhibition of TS is discussed.

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.

Inhibition of Thymidylate Synthase (TS) by the 5-Fluorouracil (5Fu) active metabolite Fluoro-deoxy-uridine-monophosphate (FdUMP) is considered to be the main mechanism of action of 5Fu. TS level from tumors and normal mucosa of 62 untreated patients who underwent surgery for primary colorectal adenocarcinoma was performed. The aim of this study was to evaluate the possibility of considering the TS level as a prognostic factor of the disease. A large variation in the level of the enzyme was found among tumors. Our data demonstrate that there is no association with age, sex, and tumor size; however there are significant relationships between TS levels and staging and histological grading. In fact the TS values are higher in Dukes' A and in G1 than in Dukes' D and G3 tumors (p0.05). Another significant association has been found between the TS level and tumor site: pts with right colon neoplasias had higher TS levels than pts with left and rectum ones. An interesting trend was found between the TS levels and survival parameters. Pts who had lower TS levels had a significantly increased risk of death (p0.05) over pts with a higher outcome. Our data support the hypothesis that a high TS level is a favourable prognostic factor in human untreated colorectal carcinomas according to our previous preliminary data (1).

5-Fluorouracil is the drug chosen for the treatment of patients with advanced colorectal carcinoma; its major site of action is thymidylate synthase (TS), resulting in pronounced and prolonged inhibition of DNA synthesis. The aim of this study was to evaluate the possibility of considering TS level in human colorectal carcinomas of previously untreated patients (pts) as a prognostic factor. Our data demonstrate that there is no association with age, sex, tumor site and tumor size; however, there is a relationship between TS levels and staging: in fact, the TS values are higher (P0.05) in Dukes-A tumors than in the others. A significant association was also found between the TS levels and survival parameters: in fact, pts with longer disease-free and overall survivals had a significantly increased TS level compared to pts with a poorer outcome (P0.05). Moreover, pts with DNA-aneuploid tumors had lower TS level (median = 0.044 pmol/mg protein) than diploid pts who had higher TS level (median = 0.093 pmol/mg protein); however the difference is not significant. Our result are based on preliminary data; however, they seem to support the hypothesis that a high TS level is a favourable prognostic factor in human colorectal carcinoma.

CD1 female mice were treated with doxorubicin (DXR, 5 mg/Kg i.v.) once a week for eight weeks or with lonidamine (LND, 100 mg/Kg i.p.) once a week for eight weeks. Other mice received lonidamine (100 mg/Kg i.p.) immediately before doxorubicin (5 mg/Kg i.v.) administration. The animals were sacrificed four weeks after the last administration and their cardiac morphology was evaluated by means of light microscopy. Lonidamine slightly reduced the extent of the atrial but not of the ventricular alterations caused by DXR. The results seem to indicate that, in this experimental model, lonidamine does not substantially modify the cardiotoxicity induced by doxorubicin.

CD 1 female mice were treated with Doxorubicin (5 mg/Kg i.v.) once a week for 8 weeks or with Isoproterenol (20 mg/Kg s.c.) once a week for 5 weeks. Other mice were treated with the chelating agent ICRF-187 (100 mg/Kg i.p.) 30 min. before Doxorubicin or Isoproterenol administration. The animals were sacrificed 4 weeks after the last administration and their cardiac morphology was evaluated by means of light microscopy. ICRF-187 significantly lessened the extent and the severity of the cardiac lesions by Doxorubicin (-68%, P less than 0.01 in left atrium; -69%, P less than 0.01 in ventricles) and the extent of those induced by Isoproterenol (-56%, P less than 0.05). These data confirm that ICRF-187 has good activity on Doxorubicin-induced myocardiopathy and provide new information about the "in vivo" effects of the compound on the cardiotoxicity caused by Isoproterenol. Moreover, they seem to confirm that a common event, probably the involvement of metal ions, plays a role in the morphologically different myocardiopathies induced by Doxorubicin or Isoproterenol.

14564 Background: Signet ring cell colorectal carcinoma ( SRCC) pure is an infrequent and highly malignant variant of colorectal cancer, while this histological component is present in 30% of all colorectal carcinomas. In our previous studies we compared the E- Cadherin, β- Catenin, Fibronectin, Ki 67 and Thymidylate Synthase (TS) expression of SRCC, the intestinal type of colorectal carcinoma (ICRC) to try to explain the pathogenesis, the aggressiveness and the low 5 Fluorouracil (5FU) responsiveness of these tumours. We found that all SRCCs had very low levels of all markers and were in the post- mitotic phase of the cell cycle. To understand their high metastatic capability we assessed the SRCC expression of Matrix Metallo Proteinase-1 (MMP1), a proteolytic enzyme, suspected to play an important role in the progression of various cancer and compared it to the ICRC one. Methods: We tested MMP1 expression immunohistochemically on formalin-fixed, paraffin-embedded samples of 32 SRCC and 70 ICRC. Differences in the distribution of the study variables, and associations between variables were assessed by means of the ?2 test. Results: SRCC showed a high expression of MMP1 over all in the invasion front of the tumour and in the neoplastic embolus rather then the ICRC (p No significant financial relationships to disclose.

The cardiac morphology of CD 1 mice undergoing two different schedules of acute (5 day) starvation and that of animals treated with a single dose (15 mg/kg i.p.) of doxorubicin, epirubicin or mitoxantrone were studied by light microscopy. Determinations of heart catalase were also carried out. Mice subjected to moderate starvation had a mean weight reduction of 18.7% and did not show heart morphological damage. A slight increase (38%) of heart catalase specific activity occurred in these animals. In animals subjected to severe starvation the weight loss was 32.2%. In this case considerable heart damage, in the form of myofibrillar loss, and a striking increase of catalase (158.5%) were seen. In the drug groups comparable weight reductions (about 15%) occurred 5 days after the treatment. Moderate heart lesions, represented by myolysis and especially by myocytic microvacuolation, were observed and appeared to be of similar degree in the 3 drug groups. Catalase specific activity increased by 119.9% in the doxorubicin animals, by 73% in the epirubicin mice and by 30.3% in the mitoxantrone ones. Light microscopy made it possible to distinguish between cardiac alterations induced by starvation and those specifically induced by antiblastics. Catalase may be helpful to indicate the existence of heart damage but it does not correlate well with the severity of the lesions by antiblastics. An additional cause of heart catalase elevation might be the free radical generation induced by the anthracyclines but not by mitoxantrone.

Experiments in mice treated with a single 10 mg/kg dose of doxorubicin (adriamycin) i.p. revealed considerable reduction in the incorporation of 3H-thymidine in DNA and of 3H-uridine in RNA, in the spleen and liver, and at mitochondrial and non-mitochondrial level in the heart. Although protein syntheses in the heart and spleen were not reduced by the drug to any great degree, they took 10 days to return to normal; conversely, liver protein syntheses were not inhibited at all and indeed presented signs of stimulation.

The importance of evaluating receptors for estrogen and progestin in human breast cancer has been pointed out by many authors. In the absence of a reference standard, receptor assays must be controlled by intra and interlaboratory quality control programs. Much interlaboratory variability exists due to non-uniform analytical protocols, non-uniform ligands, intrinsic errors and also errors in computation methods. The goals of our Italian Qualtiy Control Program on Multicenter Trials are to standardize the anaytical procedures and computation methods. Twenty Italian laboratories participated in the Quality Control Program. Each specimen was assayed for steroid receptor content according to the standardized dextran-coated-charcoal method. Data were subjected to computerized analyses by 5 different methods of calculation (Scatchard plot, direct plot, Lineweaver-Burk method, Brunauer-Emmet-Teller analysis, single-point approach). The results were than evaluated to identify intra- and inter-assay variation coefficients and to define other statistical parameters. The authors suggest different calculation methods depending on the specific experimental and/or physiopathological conditions.

In previous studies we reported that teniposide (VM26) induced acute cardiac effects in dogs seem to be related to a release of histamine and that a prior treatment with chlorpheniramine, an H, histamine blocker, prevents the onset of this phenomenon. Since histamine and other vasoactive substances also seem to be involved in doxorubicin (DXR)-induced acute cardiac effects, experiments were undertaken in the aim to prevent, as in the case of VM26, the onset of this phenomenon by administering chlorpheniramine. Since DXR-induced chronic cardiomyopathy also seems to be related to the same mechanisms involved in the onset of acute cardiac effects induced by this drug, additional studies were carried out to investigate whether a long-term treatment with VM26 could induce in mouse alterations of cardiac morphology similar to those of DXR. In addition, because the mouse is known to be extremely insensitive to histamine, further studies were performed to investigate whether DXR or VM26 administration could induce in this animal model a massive histamine release and whether a long-term treatment with high doses of histamine could elicit, similarly to DXR, alterations in cardiac morphology. The results of our experiments demonstrated that DXR (1.5 mg/kg i.v.) caused in the dog a massive histamine release and a marked impairment of cardiac inotropism. As previously described for VM26, prior treatments with chlorpheniramine completely prevented this phenomenon. Furthermore, DXR administration, at a dose level able to induce cardiac damage in the mouse (2.5 mg/kg i.v.), or that of VM26 (2 mg/kg i.v.) failed to induce a massive histamine release. In addition, long-term treatment with VM26 (2 mg/kg i.v.) or high doses of histamine (100 mg/kg i.v.), unlike DXR, did not elicit in this animal alterations of cardiac morphology. Finally, chlorpheniramine (0.15 or 0.45 mg/kg i.v.) did not prevent the onset of chronic cardiomyopathy induced by DXR in mouse. In conclusion, our results show that the role of histamine in the onset of DXR-induced chronic cardiomyopathy, at least in mouse, remains questionable and suggest that this animal, because of its high natural resistance to histamine, is not a suitable experimental model to investigate the cardiovascular pharmacology of drug-induced histamine release.

Whenever it appears impossible to modify the chemical structure of drugs with a high and established therapeutic activity but a low chemotherapeutic index, pharmacological research has to find other ways of improving the chemotherapeutic index. This problem is particularly important in the case of antitumor drugs, thus justifying research into the most suitable choice of dosage and routes of administration, as well as into the pharmacological associations which enable tumor cells to be hit at various stages of the reproductive cycle. Alternatively, the therapeutic index could be improved by the use of antagonistic compounds (like, for example, methotrexate and folinic acid) which act upon the same organic functions.

Background: Pure signet ring cell colorectal carcinoma ( SRCC) is an infrequent and highly malignant histological variant of colorectal cancer (CRC), while it is present as a histological component in colorectal carcinomas more frequently. Materials and Methods: The aim of this was to widen the knowledge of the biological factors involved in the pathogenesis and aggressiveness of SRCC by the identification and evaluation of possible molecular abnormalities. By means of immunohistochemistry the expression of the proteolytic degradation enzyme matrix metalloprotease (MMP)-1, that is a collagenase specifically degrading collagens I, II, III and of the adhesion proteins E-cadherin, B-catenin and fibronectin which are usually involved in the carcinogenesis of conventional colorectal tumours was investigated. Results: SRCCs showed a significantly greater MMP-1 expression compared to the ordinary intestinal colorectal cancer (ICRC) and a significantly reduced E-cadherin, β-catenin and fibronectin expression. Conclusions: The biological aggressiveness and strong metastatic behaviour of SRCC could be due to high MMP-1 and low expression of the adhesion molecules.

Background: Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and is the target of 5-fluorouracil (5-Fu). TS levels vary considerably among tumors and the response to 5-FU is influenced by the intratumoral activity of the enzyme, with high levels generally being associated with a poor response. Response to 5-FU also depends on TS structure and some researchers showed that variant structural forms of TS in tumour cell lines confer resistance to fluoropyrimidines so the aim our study is to analyze the whole coding regions of the TS gene to evaluate a possible mechanism of 5 Fu resistance. Materials and Methods: We performed the TS-DNA gene sequence in 68 colorectal cancer (CRC) samples from patients of different Dukes’ stages (A, B, C) and 13 metastatic samples (D). The sequence of the exons was performed amplified every exon by PCR, the amplification products of abour 180-310 bp were purified by kit Agencourt®CleanSEQ® della Beckman Coulter and sequenced by theABI3130 Genetic Analyzer della Applied Biosystems. Results. The sequencing of the 7 exons of the gene did not show any mutation in the coding region of patients at A, B, C dukes stage, while in metastatic ones was revealed a triple insertion (CCG) in the splicing region adjacent to 6 exon that could influence the splicing of the gene and as consequence the length of mRNA and the protein structure. Conclusions: 5-FU improves survival in a subgroup of CRC patients but predictive markers are required to identify patients that benefit from such a treatment, we found a mutation in the splicing region of the TS gene of metastatic CRCs that could explain their frequent 5-Fu drug resistance and a worse prognosis. A structural analysis of mRNA and protein will be necessary to valuate the real meaning of this insertion.