[Anticancer drug research in Hungary, 1950-2000]. / Daganat-kemoterápiás kutatások Magyarországon az 1950−2000 közötti években.

The present review about the history of anticancer drug research in Hungary intends to call attention to the importance of studies on their mode of action. Several lines of evidence suggest that clinically usable oncopharmacological properties could be revealed by this way. Among the numerous compounds certain alkylating sugar alcohols and 2'-deoxyuridine derivatives were submitted to detailed investigations concerning their mode of action. Myelobromol with selective action on the myeloid elements of bone marrow has been justified for its application in chronic myeloid leukemia therapy and also in bone marrow ablation before transplantation. Mitolactol is able to cross bloodbrain barrier, consequently could control certain brain tumors. 5-etil-2'-deoxyuridine by reducing dihydropyrimidine dehydrogenase activity is able to increase 5-fluorouracil concentration in the blood, resulting in improved antitumor effect. In contrast, 5-hexil-2'-deoxyuridine, as an inhibitor of glycoconjugate pathway by reducing heparan sulfate production, has the ability to prevent metastasis. Noteworthy, the remarkable effects of vinca alkaloids, antiestrogens, and GNRH analogues were also presented in this review.

Impact of SHMT1 polymorphism on the clinical outcome of patients with metastatic colorectal cancer treated with first-line FOLFIRI+bevacizumab.

The impact of thymidylate synthase (TYMS), methylenetetrahydrofolate reductase (MTHFR), and serine hydroxymethyltransferase 1 (SHMT1) gene polymorphisms and that of dihydropyrimidine dehydrogenase (DPD) enzyme activity, serum total homocysteine level, and estimated serum creatinine clearance on first-line 5-fluorouracil, leucovorin, irinotecan, and bevacizumab (FOLFIRI+bevacizumab) regimen efficacy in metastatic colorectal cancer patients was investigated. DNA was extracted from peripheral blood mononuclear cells. Genotyping was performed for TYMS 5'UTR variable number tandem repeat, TYMS 3'UTR ins/del, MTHFR C677T, and SHMT1 C1420T polymorphisms. The DPD activity of peripheral blood mononuclear cells was also determined. The univariate and multivariate analyses demonstrated that the SHMT1 1420T allele was associated with better response (P=0.025) and longer progression-free survival (PFS) (P=0.00004) and overall survival (OS) (P=0.034). Grade ≥2 hypertension was also an independent prognostic factor of longer progression-free survival and OS. Bevacizumab-related hypertension might be a predictive marker of treatment efficacy (P=0.0002 for OS) in the case of wild (CC) SHMT1 1420 genotypes only.

The association of polymorphisms in 5-fluorouracil metabolism genes with outcome in adjuvant treatment of colorectal cancer.

AIM: The purpose of this study was to investigate whether specific combinations of polymorphisms in 5-fluorouracil (5-FU) metabolism-related genes were associated with outcome in 5-FU-based adjuvant treatment of colorectal cancer. METHODS: We analyzed two cohorts of 302 and 290 patients, respectively, one cohort for exploratory analyses and another cohort for validating the exploratory analyses. A total of ten polymorphisms in genes involved in 5-FU pharmacodynamics and pharmacokinetics were studied. End points were disease-free survival (DFS) and overall survival. Multifactor dimensionality reduction was used to identify genetic interaction profiles associated with outcome. RESULTS: Low-expression alleles in thymidylate synthase (TYMS) were associated with decreased DFS and overall survival (DFS:hazard ratio [HR] exploration 2.65 [1.40-4.65]; p = 0.004, HR validation 1.69 [1.03-2.66]; p = 0.03). A specific multifactor dimensionality reduction derived combination of dihydropyrimidine dehydrogenase and TYMS polymorphisms was associated with increased DFS (HR exploration 0.69 [0.49-0.98]; p = 0.04, HR validation 0.66 [0.45-0.95]; p = 0.03). Specific combinations of functional polymorphisms in DPYD and TYMS were demonstrated to be associated with DFS and overall survival in patients receiving adjuvant 5-FU-based treatment. Specifically high TYMS expression alleles seem to be associated with decreased DFS.

Microanalytical method development for Fe, Cu and Zn determination in colorectal cancer cells.

Microanalytical methods suitable for the determination of Fe, Cu in HT-29 (human colon adenocarcinoma) cells treated with different iron compounds (Fe(II) sulfate, Fe(III) chloride, Fe(III) citrate and Fe(III) transferrin) and cultured in medium supplemented or not with 10% (v/v) fetal calf serum (FCS) by total reflection X-ray fluorescence spectrometry (TXRF) and simultaneous graphite furnace atomic absorption spectrometry (GF-AAS) were developed. The developed TXRF method was also suitable for Zn determination in the samples. The main advantage of the proposed methods is the execution of all sample preparation steps following incubation and prior to the elemental analysis in the same Eppendorf tubes. Sample preparation was performed at microscale (115 µL sample volume) with 65% nitric acid and 30% hydrogen peroxide. According to scanning electron microscopic measurements, the organic matrix of the cell samples could be eliminated to the extent that accurate results were obtained for Cu and Fe by analyzing the same samples by TXRF and GF-AAS. Concerning the iron uptake, HT-29 cells incubated in FCS-free medium contained Fe in cca. 5-50 times higher amounts compared to cells cultured in FCS supplemented medium. Pronounced differences in the iron uptake compared to the iron supply (inorganic vs. organic chelated as well as iron(II) vs. iron(III)) were observed in the case of cell lines incubated in FCS-free medium.

SHMT1 1420 and MTHFR 677 variants are associated with rectal but not colon cancer.

BACKGROUND: Association between rectal or colon cancer risk and serine hydroxymethyltransferase 1 (SHMT1) C1420T or methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms was assessed. The serum total homocysteine (HCY), marker of folate metabolism was also investigated. METHODS: The SHMT1 and MTHFR genotypes were determined by real-time PCR and PCR-RFLP, respectively in 476 patients with rectal, 479 patients with colon cancer and in 461 and 478, respective controls matched for age and sex. Homocysteine levels were determined by HPLC kit. The association between polymorphisms and cancer risk was evaluated by logistic regression analysis adjusted for age, sex and body mass index. The population stratification bias was also estimated. RESULTS: There was no association of genotypes or diplotypes with colon cancer. The rectal cancer risk was significantly lower for SHMT1 TT (OR = 0.57, 95% confidence interval (CI) 0.36-0.89) and higher for MTHFR CT genotypes (OR = 1.4, 95%CI 1.06-1.84). A gene-dosage effect was observed for SHMT1 with progressively decreasing risk with increasing number of T allele (p = 0.014). The stratified analysis according to age and sex revealed that the association is mainly present in the younger (< 60 years) or male subgroup. As expected from genotype analysis, the SHMT1 T allele/MTHFR CC diplotype was associated with reduced rectal cancer risk (OR 0.56, 95%CI 0.42-0.77 vs all other diplotypes together). The above results are unlikely to suffer from population stratification bias. In controls HCY was influenced by SHMT1 polymorphism, while in patients it was affected only by Dukes' stage. In patients with Dukes' stage C or D HCY can be considered as a tumor marker only in case of SHMT1 1420CC genotypes. CONCLUSIONS: A protective effect of SHMT1 1420T allele or SHMT1 1420 T allele/MTHFR 677 CC diplotype against rectal but not colon cancer risk was demonstrated. The presence of SHMT1 1420 T allele significantly increases the HCY levels in controls but not in patients. Homocysteine could be considered as a tumor marker in SHMT1 1420 wild-type (CC) CRC patients in Dukes' stage C and D. Further studies need to clarify why SHMT1 and MTHFR polymorphisms are associated only with rectal and not colon cancer risk.

Enhanced 5-fluorouracil cytotoxicity in high cyclooxygenase-2 expressing colorectal cancer cells and xenografts induced by non-steroidal anti-inflammatory drugs via downregulation of dihydropyrimidine dehydrogenase.

PURPOSE: To prove that 5-FU cytotoxicity could be increased by combination with low-dose non-steroidal anti-inflammatory drugs (NSAIDs) (indomethacin or NS-398) in high cyclooxygenase-2- (COX-2) expressing cells and xenografts through the modulation of dihydropyrimidine dehydrogenase (DPD) mRNA expression and/or enzyme activity. METHODS: HT-29 cells were grown on collagen IV coated plates (HT-29-C). The antiproliferative effect of 5-fluorouracil (5-FU) +/- NSAIDs was examined on non-COX-2 expressing HT-29 and COX-2-expressing HT-29-C cells by sulphorhodamine B assay. The COX-2 and DPD expressions were visualized by immunofluorescent staining, and prostaglandin E(2) levels were measured by ELISA kit. The HT-29 xenograft was established in SCID mice and treated with 5-FU +/- NSAIDs for 5 days. The tumor volume, enzyme activity, and DPD mRNA expression were investigated by caliper, radioenzymatic method, and real-time RT-PCR, respectively. The drug interaction was calculated for both combinations (5-FU + indomethacin and 5-FU + NS-398). RESULTS: Collagen IV up-regulated significantly the COX-2 and DPD mRNA, and protein expressions, and also their enzyme activities in HT-29 cells. NSAIDs enhanced in a synergistic manner the cytotoxic effect of 5-FU treatment both in vitro and in vivo. Downregulation of DPD was observed after 5-FU monotherapy, but the combined effect of NSAIDs and 5-FU on DPD mRNA expression, and enzyme activity was superior to the effect of 5-FU alone. CONCLUSIONS: Since 5-FU + NSAID treatment can alter the DPD enzyme activity resulting in an enhanced cytotoxic effect, further studies in clinical practice are warranted.

Co-inhibition of cyclooxygenase-2 and dihydropyrimidine dehydrogenase by non-steroidal anti-inflammatory drugs in tumor cells and xenografts.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) may be able to enhance the antitumor effect of cancer drugs. Cyclooxygenase-2 (COX-2) is the best characterized target of NSAIDs. It was demonstrated that elevated dihydropyrimidine dehydrogenase (DPD) and COX-2 activities influence the response to 5-fluorouracil (5-FU). We previously showed that NSAIDs increased 5-FU sensitivity only in high COX-2-expressing cancer cells. MATERIALS AND METHODS: The effect of indomethacin and NS-398 on DPD activity and mRNA expression in a high COX-2-expressing (determined by Western blotting, immunoflourescence and immunohistochemistry) cell line (24-, 48-hour, 10-day treatment) and xenograft (3-week treatment) was investigated. RESULTS: The coexistence of high COX-2 and DPD activity or low activities of both enzymes were detected. After treatment with NSAIDs, a simultaneous and significant decrease of both activities was also demonstrated. CONCLUSION: NSAIDs could be promising modulators of fluorouracil-based chemotherapy, especially in high COX-2-expressing tumours.

Enhancement of 5-fluorouracil efficacy on high COX-2 expressing HCA-7 cells by low dose indomethacin and NS-398 but not on low COX-2 expressing HT-29 cells.

The antiproliferative effect of 5-fluorouracil (5-FU) in the presence of low dose non-steroidal anti-inflammatory drugs (NSAIDs) on high cyclooxygenase-2 (COX-2)-expressing HCA-7 and low COX-2-expressing HT-29 colon carcinoma cell lines was investigated. Pharmacogenetic parameters were studied to characterize the 5-FU sensitivity of the two cell lines. Thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) polymorphisms were determined by PCR analysis. Cell proliferation was measured by SRB assay, cell cycle distribution and apoptosis by FACS analysis. Cyclooxygenase expression was detected by Western blot and also by fluorescence microscopy. Prostaglandin E(2) (PGE(2)) levels were investigated with ELISA kit. The HT-29 cell line was found to be homozygous for TS 2R and 1494ins6 and T homozygous for MTHFR 677 polymorphisms predicting high 5-FU sensitivity (IC(50): 10 microM). TS 3R homozygosity, TS 1496del6 and MTHFR 677CT heterozygosity may explain the modest 5-FU sensitivity (IC(50): 1.1 mM) of the HCA-7 cell line. Indomethacin and NS-398 (10 microM and 1.77 microM, respectively) reduced the PGE(2) level in HCA-7 cells (>90%). Low concentrations of NSAIDs without antiproliferative potency increased the S-phase arrest and enhanced the cytotoxic action of 5-FU only in HCA-7 cells after 48-hours treatment. The presented data suggested that the enhancement of 5-FU cytotoxicity by indomethacin or NS-398 applied in low dose is related to the potency of NSAIDs to modulate the cell-cycle distribution and the apoptosis; however, it seems that this effect might be dependent on cell phenotype, namely on the COX-2 expression.

Toxic encephalopathy and delayed MTX clearance after high-dose methotrexate therapy in a child homozygous for the MTHFR C677T polymorphism.

BACKGROUND: High-dose methotrexate (HD-MTX) is one of the most important agents in the therapy of osteosarcoma (OSC). Acute and delayed toxicities still constitute clinical problems. Methylenetetrahydrofolate reductase (MTHFR) has a key role in the folate cycle. In case of homozygosity of the 677C-->T polymorphism, treatment with antimetabolites, such as MTX, can cause additional toxicity. CASE REPORT: In the present work, we describe the case of a 10-year-old boy with OSC. After the first HD-MTX infusion (12 g/m2/6 h) acute neurological disturbances were detected followed by severe hepatotoxicity. Plasma concentrations of MTX and 7-OH-MTX showed delayed clearance. Calcium folinate was administered to the patient until +186 hours. Tha patient was homozygous for the 677 polymorphism and wild-type for the 1298 polymorphism of the MTHFR gene. CONCLUSION: We hypothesize that MTX toxicity can be explained by the association between homozygosity of the MTHFR C677T polymorphism causing disturbances in the folate status and thus an enhanced vulnerability of the central nervous system to antimetabolites and to the prolonged MTX exposure due to delayed MTX clearance.

[Efforts to enhance the efficiency of tumor chemotherapy]. / Törekvések a daganatkemoterápia hatékonyságának fokozására.

Antiproliferative cytotoxic agents, of which several are still in clinical practice nowadays, could be characterized by low selectivity, narrow therapeutic index, medium or serious side effects and rapid formation of resistance. In the limited efficacy of these drugs several factors are playing a role such as the age, gender and pharmacogenetics of the patients, the morphological and biological feature of the tumor, moreover, pharmacokinetics and pharmacodynamics of the drugs. The question could be justified if there are methods which, by influencing the above parameters, are helpful in enhancing the efficacy and decreasing the toxic side effects of these drugs. Since a long time we have been interested in evaluating methods of preclinical and clinical level for increasing drug efficacy. The aim of this minireview is to give a short summary about our previous and present projects aiming: 1.) to characterize and mitigate toxic side effects of several cytotoxic agents; 2.) to decrease the toxic side effects and improve the antitumor effect of 5-fluorouracil by biochemical modulation and 3.) to study the possibility of individualized drug selection, based on the pharmacobiochemical and pharmacogenetic characteristics of the patients.

[Pharmacogenetic studies on the prediction of efficacy and toxicity of fluoropyrimidine-based adjuvant therapy in colorectal cancer]. / Farmakogenetikai vizsgálatok a fluoropirimidint tartalmazó adjuváns kezelés hatékonyságának és toxicitásának elôrejelzésére colorectalis daganatokban.

The cytotoxic effect of 5-fluorouracil (5-FU) is mediated by the inhibition of thymidylate synthase (TS), however, at the same time 5-FU is catabolized by dihydropyrimidine dehydrogenase (DPD). Efficacy of 5-FU may therefore depend on the TS and DPD activity and on pharmacogenetic factors influencing these enzymes. Our aims were (1) to determine the distribution of DPD activity, the frequency of DPD deficiency and the DPD (IVS14+1G>A) mutation in the peripheral blood mononuclear cells of colorectal cancer (CRC) patients, and study the relationship between DPD deficiency and toxicity of 5-FU; (2) to investigate the influence of TS polymorphisms and DPD activity on the survival of CRC patients receiving 5-FU-based adjuvant therapy. The frequency of DPD deficiency was determined by radiochemical methods in the peripheral blood mononuclear cells (PBMCs) of 764 CRC patients treated with 5-FU. The relationship between the TS polymorphisms, DPD activity and the disease-free and overall survival was studied in 166 CRC patients receiving 5-FU-based adjuvant therapy. TS polymorphisms were determined in the DNA samples separated from the PBMCs, by PCR-PAGE and PCR-RFLP-PAGE (restriction fragment length polymorphism) methods. Low DPD values (<10 pmol/min/106 PBMCs) were demonstrated in 160/764 patients (20.9%), and of those DPD deficiency (<5 pmol/min/106 PBMCs) was verified in 38 patients (4.9%). In the latter group severe (>Gr 3) toxicity was found in 87%. The prevalence of the DPD IVS14+1G>A mutation among the 38 DPD-deficient patients was 7.8% (3/38) and was accompanied by severe Gr 4 toxic symptoms (neutropenia, mucositis, diarrhea). TS polymorphisms showed a relationship with the survival of CRC patients. It is important to mention that by combining the 3-3 genotypes of 5'-TSER and 3'-TSUTR polymorphisms the obtained 8 genotype combinations showed significantly different Kaplan-Meier survival curves. The evaluation of these curves with Cox regression analysis resulted in two prognostically different groups: "A" good prognosis (RR<1) and "B" bad prognosis (RR>1). The disease-free- and overall survival of these two groups were significantly different. DPD activity also showed correlation with the survival; patients with DPD activity <10 pmol/min/106 PBMCs showed significantly longer disease-free and overall survival. The determination of DPD activity proved to be a more valuable parameter in the evaluation of serious 5-FU-related toxicity compared to the IVS14+1G>A mutation analysis. According to the Cox multivariate analysis the combination of germline TS polymorphisms and DPD activity is/an independent prognostic marker of survival in CRC patients treated with adjuvant 5-FU therapy.

Influence of thymidylate synthase gene polymorphisms on the survival of colorectal cancer patients receiving adjuvant 5-fluorouracil.

The present study aimed to prospectively investigate the influence of thymidylate synthase (TS) polymorphisms (5'-TSER, 3'-TSUTR) on the disease-free survival (DFS) and overall survival (OS) of patients with colorectal cancer (CRC) who were treated with adjuvant 5-fluorouracil (5-FU) therapy. Patients were followed up for 19+/-14 months (median+/-SD). TS genotypes were determined from the peripheral blood mononuclear cells of 166 patients by polymerase chain reaction-polyacrylamide gel electrophoresis and restriction fragment length polymorphism methods. 5'-TSER 3R homozygotes showed significantly longer DFS (P = 0.048) and OS (P = 0.009). The 5'-TSER and 3'-TSUTR genotype combination groups showed a significant difference for DFS (P = 0.039) and OS (P = 0.029). Significantly better DFS (P = 0.049) and OS (P = 0.043) were observed for 6 bp/6 bp genotypes in 5'-TSER heterozygotes (n = 80). Based on this, and on hazard ratios obtained by Cox regression analysis of the DFS of genotype-combinations, the patients were classified as belonging to prognostic groups A and B. The DFS and OS of these two groups showed a highly significant difference (P = 0.002 and 0.001). In the multivariate Cox regression model, beside tumour location, the prognostic classification (groups A and B) proved to be an independent prognostic factor. Our data suggest that those TS genotypes and their combinations (group A: 3R/3R with any 3'-TSUTR genotype and 2R/3R with 6 bp/6 bp), which have been reported earlier as having high TS expression, predict significantly longer DFS and OS. We found that a combination of germline TS polymorphisms is an independent prognostic marker in selecting CRC patients with worse prognosis, and it may be worthwhile to examine whether these patients would benefit from an alternative therapy.

[Potentiation of 5-fluorouracil efficacy. Molecular mechanisms playing a role in the cytotoxic action of 5-fluorouracil and 5-ethyl-2'-deoxyuridine (EUdR) combination]. / Az 5-fluorouracil (5-FU) hatékonyságának fokozása. Az 5-FU és 5-etil-2'-dezoxiuridin (EUdR) kombináció hatásában szerepet játszó molekuláris tényezôk.

UNLABELLED: Pharmacologic modulation of 5-fluorouracil (5-FU) metabolism provides a possibility for the enhancement of its clinical efficacy. AIM: The purpose of the present work was to study the effect of 5-ethyl-2'-deoxyuridine (EUdR), a potent 5-FU modulator, on different molecular mechanisms, influenced by 5-FU itself, and to obtain further data about the mode of action of the combination. MATERIALS AND METHODS: SW620 cell line was used for the experiments. Cytotoxicity was studied by MTT test, cell kinetic changes by FACStar flow cytometer, apoptosis by fluorescent microscope after staining the cells with acridine orange and ethydium bromide, DNA fragmentation by PAGE electrophoresis after RNase and proteinase-K digestion, thymidine incorporation with 3H-thymidine, p53 and PCNA protein expression by Western blotting. RESULTS: The cytotoxicity of 5-FU was potentiated dose dependently by EUdR. One hundred muM concentration of EUdR resulted in a 40% decrease of the IC50 value of 5-FU. Cell cycle arrest in the G2/M transition phase was most pronounced after combined treatment with 5-FU+EUdR. EUdR potentiated the incorporation of 3Hthymidine into DNA. In addition to the increase of apoptosis rate, the expression of p53 protein, caused by 5-FU was further potentiated by UdR. CONCLUSION: This study demonstrated a potential novel approach to increase the efficacy of 5-FU by EUdR, which incorporated two complementary molecular actions, the selective modulation of TS inhibition and potentiation of the p53 protein expression, consequently leading to an increase in the apoptotic rate.

[The clinical importance of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in the 5-fluoropyrimidine-based therapy of metastatic colorectal tumours]. / A metiléntetrahidrofolát-reduktáz (MTHFR) C677T polimorfizmus klinikai jelentôsége a metasztatikus colorectalis daganatok 5-fluoropirimidin-alapú kezelésében.

The authors investigated the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in 101 metastatic colorectal cancer patients treated with 5-fluoropyrimidine-based therapy and in 196 healthy individuals by PCR-RFLP method. There was no significant difference in genotype distribution of patients and healthy controls, and between subgroups investigated according to clinical parameters (age, gender, tumor location, grade and treatment type). However, after a 3-30 (median 18.5) months follow-up the survival of patients with T allele proved to be better than that of patients with wild type (CC) genotype (p=0.036). In case of CT and TT genotypes the survival of patients receiving only first line therapy was significantly shorter than that of patients receiving more lines of treatment (p=0.015). Determination of MTHFR C677T polymorphism has prognostic value in case of patients with metastatic colorectal cancer receiving 5-fluoropyrimidine-based therapy, and may help in designing the individual (group) tailored therapy.

Heterozygote deficiency in thymidylate synthase enhancer region polymorphism genotype distribution in Hungarian colorectal cancer patients.

Thymidylate synthase (TS) gene polymorphisms are important as prognostic factors in cancer chemotherapy, but recent results describe that the TS enhancer region (TSER) polymorphic genotypes may also modulate risk for malignancies. Two functionally important and ethnically diverse polymorphisms are present on the TS transcript, TSER, a repeat polymorphism (2 or 3 repeats; 2R, 3R) affecting TS expression, and a 6 bp ins/del polymorphism on the 3' UTR (position TS1494, del6 or ins6), which may influence mRNA stability. Hungarian population has one of the highest colorectal cancer (CRC) mortality rates in Europe, and several elevated dietary risk factors affect a large part of the population. In our study (99 primary CRC cases), population analysis of the patient group genotype frequencies revealed a departure from the Hardy-Weinberg equilibrium and significant heterozygote deficiency (p < 0.05) at the TSER locus. Despite the strong linkage between the 2 polymorphic loci, case TS1494del genotype frequencies were normally distributed, as well as the genotype frequencies of the healthy control population (n = 102), at both loci. Case-control comparison demonstrated a lower relative risk of TSER heterozygotes (OR = 0.47; CI = 0.27-0.83; p = 0.008) and a possible higher prevalence of the 3R3R&ins6/del6 in the CRC group. The observation that heterozygotes are those less susceptible for CRC in the Hungarian population may support the possibility of 2 different pathways in which TS may play a role in colorectal carcinogenesis, probably nutrient (or folate)-dependently. The lack of similar genotype effect seen with TS1494del polymorphism and the increased presence of one genotype combination (3R3R&ins6/del6) in the patient group suggest a possible TS haplotype effect influencing CRC risk.

Healing of esophageal anastomoses performed with the biofragmentable anastomosis ring versus the end-to-end anastomosis stapler: comparative experimental study in dogs.

The biofragmentable anastomosis ring (BAR) has been used successfully for anastomoses from the stomach to the upper rectum. The healing of intrathoracic esophageal anastomoses performed with the BAR or an end-to-end anastomosis (EEA) stapler on an experimental model was compared. Parameters of tissue repair were evaluated: macroscopic examination, bursting strength (BS), collagen (hydroxyproline, or HP), histology (H&E and Picrosirius red staining for collagen). A series of 48 mongrel dogs were randomly separated into two groups (30 BAR, 18 stapler) and subgroups according to the time of autopsy (days 4, 7, 14, 28). Mortality was 13.3% (4 BAR cases) with two deaths not related to surgery (excluded). There were four leaks in the BAR group (14.3%) and no leaks or deaths but two strictures in the stapler group. BS was significantly higher in the BAR group during the first week, and values were almost equal from the second week with both methods. The HP rate was significantly reduced on days 4 and 7 in both groups compared to the reference values; the values were close to reference values from the second week (lower in the BAR group). Stapled anastomoses caused less pronounced inflammation and were associated with an earlier start of regeneration, but the difference was not significant compared to that in the BAR group. Accumulation of new collagen (green polarization) started on day 7 in both groups, but maturation (orange-red polarization) was significantly more advanced in the BAR group after the second week. A strong linear correlation between the BS and HP rate was found with both methods. There was no significant difference in the complication rate or healing of intrathoracic BAR and stapled anastomoses. The BAR method is simple, quick, and safe; and it seems to be a feasible procedure for creating intrathoracic esophageal anastomoses in dogs.

Prognostic significance of the thymidylate biosynthetic enzymes in human colorectal tumors.

The aim of the present study was to determine the relative intratumoral activity of two pyrimidine biosynthetic enzymes, i.e. thymidylate synthase (TS) and thymidine kinase (TK), in human colorectal cancers to compare their possible relationship with demographic and pathologic characteristics of the patients and their tumors, and moreover to evaluate their predictive significance regarding 5-fluorouracil (5-FU) sensitivity and the overall survival of patients, respectively. TS and TK levels were significantly increased in the tumor compared to peritumoral tissue. However, no significant relationship between TS/TK activity and demographic features of the patients or pathologic characteristics of their tumors could be demonstrated. Kaplan-Meier analysis showed that the overall survival of patients with low TS activity was significantly longer (p < 0.012) compared to those with high TS activity. Such a difference could not be demonstrated between patients with high or low TK activity; however, combined evaluation of the two parameters proved that TK may contribute to the more precise assessment of disease prognosis, and it may further influence treatment decisions, i.e. the selection of patients for adjuvant therapy with 5-FU and folinic acid. Multivariate analysis showed that among the variables tested, beside Dukes' stage, TS and TK activities were significant prognostic factors for the overall survival of colorectal cancer patients.