Judit Kralovánszky

Putative role of dihydropyrimidine dehydrogenase in the toxic side effect of 5-fluorouracil in colorectal cancer patients.

Dihydropyrimidine dehydrogenase (DPD) is the first and rate limiting enzyme in the catabolism of 5-fluorouracil (5-FU). It has been reported from various laboratories that the plasma concentration of 5-FU was influenced by DPD activities in various normal human organs (e.g. liver or lymphocytes). Since the congenital deficiency in DPD caused severe, in some cases lethal, FU-related toxicity, it was decided to collect data about the DPD activity in colorectal cancer patients in order to investigate the possible correlation between the enzyme activity and appearance of the side effects of 5-FU. Assuming that DPD activity in lymphocytes represents the 5-FU catabolic capacity of the organism, DPD activity was determined in the lymphocytes of 48 patients with colorectal cancer after surgery during the therapeutic course with 5-FU and folinic acid. On the basis of the enzyme activity, patients were divided into three categories: low (DPD <5.03 pmol/min/106 lymphocytes); medium (DPD = 5.04–13.25 pmol/min/106 lymphocytes), and high (DPD > 13.26 pmol/min/106 lymphocytes) activity groups. By evaluating the toxic side effects during the 5-FU + folinic acid treatment, the following results were obtained. In the low DPD activity group, 9 of 11 patients had 5-FU-related side effects (mucositis, diarrhea, myelotoxicity, angina pectoris, hypertension). In 3 patients, no change of the therapy was needed, in 3 patients symptoms could be reversed by dose reduction of 5-FU while in 3 patients interruption of 5-FU therapy was needed. In the medium DPD activity group, mild toxicity (diarrhea, transitory hypertension) occurred in 5 of 29 and in the high activity group (diarrhea) in 1 of 8 patients, respectively. In these last two groups, no dose reduction of 5-FU was necessary. The present study furnished further evidence for the possible correlation between the 5-FU side effects and DPD function. Consequently, it is recommended to measure DPD activity prior to 5-FU based chemotherapy, which might be helpful in avoiding drug-related toxicity by adjusting the dose of 5-FU individually.

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.

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.

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.

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.

5-Ethyl-2'-deoxyuridine, a modulator of both antitumour action and pharmacokinetics of 5-fluorouracil

Abstract The aim of the present studies was to elucidate the effects and optimal modulatory conditions of 5-ethyl-2′-deoxyuridine (EtdUrd) on the antitumour efficacy, pharmacokinetics and catabolism of 5-fluorouracil (5-FU) on Colon-26 and Colon-38 murine tumours. HPLC and GC-MS techniques were used to measure the concentrations of 5-FU, dihydro-5-fluorouracil, EtdUrd, 5-ethyluracil and uridine in the plasma and that of 5-FU and 5-fluoro-2′-deoxyuridine monophosphate (FdUMP) in the tumours. It was shown that EtdUrd, given 1 h before 5-FU, selectively enhanced the antitumour action of 5-FU, without significantly increasing its toxic side-effects, thus resulting in an approximately three times higher therapeutic index. Pharmacokinetic studies revealed that 1 h after 400 mg/kg EtdUrd administration – i.e. at the time of 5-FU treatment – the plasma concentration of EtdUrd was 269 μM, and that of 5-ethyluracil, as the major metabolite of EtdUrd, was 421 μM. It is of interest that EtdUrd pretreatment did not change the maximal plasma concentration of 5-FU; however, the half-life of the terminal elimination increased from 114.5 min to 171.2 min and thus the mean residence time of 5-FU rose significantly (P < 0.05). After the combined treatment, the maximal concentration of dihydro-5-fluorouracil in the plasma decreased from 61.06 μM to 29.70 μM (P < 0.01). The intratumoral concentrations of 5-FU were 34%–158% higher 6–96 h after the combined treatment than after the single 5-FU treatment. EtdUrd also caused a moderate increase in the intratumoral level of FdUMP. It is noteworthy, that EtdUrd increased the endogenous uridine concentration in the plasma from 18 μM to a maximum of 249 μM, and the level remained high for longer than 6 h. The present studies indicate that EtdUrd enhances the therapeutic index of 5-FU by reducing the catabolism, prolonging the plasma and intratumoral concentrations of 5-FU and, at the same time, offering protection to normal organs by increasing the endogenous uridine level.

Alkaline phosphatase activity in human and rat liver tumors.

Enzyme activity measurements of alkaline phosphatase in surgically removed human liver tumors showed elevated level of the enzyme in 6 focal nodular hyperplasias, reduction in 8 primary hepatocellular carcinomas, and no change in the 4 adenoma samples. The activity represented liver type of alkaline phosphatase nearly in all cases because it could be inhibited by L-homoarginine more extensively than by L- phenylalanine. Studies on polyacrylamide gel electrophoresis indicated the presence of a variant type isoenzyme only in one focal nodular hyperplasia and in two hepatocellular carcinomas, one of which showed a fibrolamellar structure whereas the other was associated to cirrhosis. The importance of the elevated amount of connective tissue in the tumor, resulting in an isoenzyme shift of alkaline phosphatase, received substantial support upon comparing chemically induced rat liver tumors with and without cirrhosis.

Placental Type Alkaline Phosphatase in Possibly Premalignant Alterations of Human Gastric Mucosa

The occurrence of placental type alkaline phosphatase (Regan enzyme) was studied in various pathological conditions of the human stomach using polyacrylamide gel electrophoresis and various inhibition techniques. Placental type alkaline phosphatase could be demonstrated in 16 out of 22 gastric carcinomas, in 3 out of 11 moderate and 6 out of 6 severe dysplasias, we failed however to demonstrate it in any other pathological conditions of the stomach, i.e. in intestinal metaplasia. There was no correlation between the occurrence of Regan enzyme and histological type of gastric carcinomas. The appearance of placental type alkaline phosphatase in gastric dysplasias may be suggestive of malignant transformation and its determination in repeated biopsy samples may be a help in the follow-up of patients with abnormal gastroscopic findings.

Efforts to enhance the efficiency of tumor chemotherapy

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.