Zbigniew Banaszkiewicz

Persistent oxidative stress in colorectal carcinoma patients

We examine whether the level of 8‐oxo‐2′‐deoxyguanosine (8‐oxodGuo) in lymphocytes DNA is higher in colon cancer when compared to the control group. Factors that may influence oxidative stress such as antioxidant vitamins and uric acid were also determined. Blood samples were obtained from a control group of 55 healthy persons and 43 colon cancers. 8‐OxodGuo level and the vitamins concentration were measured by high‐performance liquid chromatography. The levels of 8‐oxodGuo were significantly higher whereas the concentrations of the vitamins and uric acid were significantly lower in colon cancer patients than in control group. Therefore, the decreased concentration of antioxidant vitamins together with lower amount of uric acid may be responsible for the formation of pro‐oxidative environment in blood of colorectal carcinoma patients.

Germline MSH2 and MLH1 mutational spectrum including large rearrangements in HNPCC families from Poland (update study).

Germline mutations in the DNA mismatch repair genes MSH2 and MLH1 account for a significant proportion of hereditary non‐polyposis colorectal cancer (HNPCC) families. One approach by which development of an efficient DNA‐testing procedure can be implemented is to describe the nature and frequency of common mutations in particular ethnic groups. Two hundred and twenty‐six patients from families matching the Amsterdam II diagnostic criteria or suspected HNPCC criteria were screened for MSH2 and MLH1 germline mutations. Fifty different pathogenic mutations were found, 25 in MSH2 and 25 in MLH1. Twenty‐four of these had not previously been described in other populations. Among our 78 families with MSH2 or MLH1 mutations, 54 (69.2%) were affected by recurrent mutations including 38 found at least twice in our own series. Two of the most frequent alterations were a substitution of A to T at the splice donor site of intron 5 of MSH2 and a missense change (A681T) of MLH1 found in 10 and eight families, respectively. Among large deletions detected by the multiplex ligation‐dependent probe amplification assay, exon 9 deletions in the MSH2 gene were found in two families. Our results indicate that a screening protocol specific for the Polish population that is limited to the detection of all reported mutations will result in the identification of the majority of changes present in MLH1 and MSH2 genes in Polish HNPCC kindreds.

Variant alleles of the CYP1B1 gene are associated with colorectal cancer susceptibility

BackgroundCYP1B1 is a P450 enzyme which is involved in the activation of pro-carcinogens to carcinogens as well as sex hormone metabolism. Because differences in the activity of the enzyme have been correlated with variant alleles of single nucleotide polymorphisms (SNPs), it represents an attractive candidate gene for studies into colorectal cancer susceptibility.MethodsWe genotyped 597 cancer patients and 597controls for three CYP1B1 SNPs, which have previously been shown to be associated with altered enzymatic activity. Using the three SNPs, eight different haplotypes were constructed. The haplotype frequencies were estimated in cases and controls and then compared. The odds ratio for each tumour type, associated with each haplotype was estimated, with reference to the most common haplotype observed in the controls.ResultsThe three SNPs rs10012, rs1056827 and rs1056836 alone did not provide any significant evidence of association with colorectal cancer risk. Haplotypes of rs1056827 and rs10012 or rs1056827 and rs1056836 revealed an association with colorectal cancer which was significantly stronger in the homozygous carriers. One haplotype was under represented in the colorectal cancer patient group compared to the control population suggesting a protective effect.ConclusionGenetic variants within the CYP1B1 that are associated with altered function appear to influence susceptibility to a colorectal cancer in Poland. Three haplotypes were associated with altered cancer risk; one conferred protection and two were associated with an increased risk of disease. These observations should be confirmed in other populations.

8-Oxo-7,8-dihydroguanine and uric acid as efficient predictors of survival in colon cancer patients.

The aim of this work was to answer the question whether the broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair are appropriate prognosis factors of colon cancer (CRC) patients survival? The following parameters were analyzed for 89 CRC patients: concentration of uric acid and vitamins A, E, C in plasma; levels of 8‐oxodGuo (8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine) in DNA of leukocyte and colon tissues; urinary excretion rates of 8‐oxodGuo and 8‐oxoGua (8‐oxo‐7,8‐dihydroguanine); the activity and mRNA or protein level of repair enzymes OGG1, APE1, ANPG, TDG and PARP1. All DNA modifications and plasma antioxidants were analyzed using high performance liquid chromatography (HPLC) or HPLC/gas chromatography‐mass spectrometry techniques. Expression of repair proteins was analyzed by QPCR, Western or immunohistochemistry methods. Longer survival coincided with low levels of 8‐oxodGuo/8oxoGua in urine and 8‐oxodGuo in DNA as well as with high concentration of uric acid plasma level. In contrast to expectations, longer survival coincided with lower mRNA level in normal colon tissue of the main 8‐oxoGua DNA glycosylase, OGG1, but no association was found for PARP‐1 expression. When analyzing simultaneously two parameters the discriminating power increased significantly. Combination of low level of urinary 8‐oxoGua together with low level of 8‐oxodGuo in leukocyte (both below median value) or high concentration of plasma uric acid (above median value) have the best prediction power. Since prediction value of these parameters seems to be comparable to conventional staging procedure, they could possibly be used as markers to predict clinical success in CRC treatment.

PARP-1 expression is increased in colon adenoma and carcinoma and correlates with OGG1.

The ethiology of colon cancer is largely dependent on inflammation driven oxidative stress. The analysis of 8-oxodeoxyguanosine (8-oxodGuo) level in leukocyte DNA of healthy controls (138 individuals), patients with benign adenomas (AD, 137 individuals) and with malignant carcinomas (CRC, 169 individuals) revealed a significant increase in the level of 8-oxodGuo in leukocyte DNA of AD and CRC patients in comparison to controls. The counteracting mechanism is base excision repair, in which OGG1 and PARP-1 play a key role. We investigated the level of PARP-1 and OGG1 mRNA and protein in diseased and marginal, normal tissues taken from AD and CRC patients and in leukocytes taken from the patients as well as from healthy subjects. In colon tumors the PARP-1 mRNA level was higher than in unaffected colon tissue and in polyp tissues. A high positive correlation was found between PARP-1 and OGG1 mRNA levels in all investigated tissues. This suggests reciprocal influence of PARP-1 and OGG1 on their expression and stability, and may contribute to progression of colon cancer. PARP-1 and OGG1 proteins level was several fold higher in polyps and CRC in comparison to normal colon tissues. Individuals bearing the Cys326Cys genotype of OGG1 were characterized by higher PARP-1 protein level in diseased tissues than the Ser326Cys and Ser326Ser genotypes. Aforementioned result may suggest that the diseased cells with polymorphic OGG1 recruit more PARP protein, which is necessary to remove 8-oxodGuo. Thus, patients with decreased activity of OGG1/polymorphism of the OGG1 gene and higher 8-oxodGuo level may be more susceptible to treatment with PARP-1 inhibitors.

CHEK2 mutations and HNPCC-related colorectal cancer

Recently, the 1100delC variant of cell cycle checkpoint kinase 2 (CHEK2) has been reported to confer a colorectal cancer risk in hereditary non‐polyposis‐colorectal cancer (HNPCC) and HNPCC‐related families in the Netherlands. To investigate whether CHEK2 mutations confer increased cancer risk in HNPCC and HNPCC‐related families in Poland, we genotyped 463 probands from HNPCC and HNPCC‐related families, and 5,496 controls for 4 CHEK2 alleles (1100delC, IVS2+1G>A, del5395, I157T). All 463 probands were screened for mutations in the HNPCC‐related genes MSH2, MLH1 and MSH6. A positive association was observed for HNPCC‐related cancer and the I157T missense CHEK2 mutation (OR = 1.7; p = 0.007), but not for the truncating alleles (OR = 1.0; p = 1.0). The association with the I157T was seen both for the 117 cases who fulfill Amsterdam criteria (OR = 1.9; p = 0.1) and for the 346 cases who do not fulfill the criteria (OR = 1.6; p = 0.03). One hundred forty‐five of the 463 families had a mutation in MSH2, MLH1 or MSH6 (MMR‐positive families). A positive association between the CHEK2 I157T mutation and HNPCC‐related cancer was observed only for MMR‐negative cases (OR = 2.1; p = 0.0004), but not for MMR‐positive cases (OR = 0.8; p = 0.9). The association with I157T was particularly strong for MMR‐negative cases with familial colorectal cancer (2 or more first‐degree relatives affected) (OR = 2.5; p < 0.0001). We conclude that the I157T variant of CHEK2 increases the risk of colorectal cancer among MMR‐negative, HNPCC/HNPCC‐related families in Poland.

Heteroplasmic substitutions in the entire mitochondrial genomes of human colon cells detected by ultra-deep 454 sequencing

Mitochondrial DNA (mtDNA) heteroplasmy has been widely described from clinical, evolutionary and analytical points of view. Historically, the majority of studies have been based on Sanger sequencing. However, next-generation sequencing technologies are now being used for heteroplasmy analysis. Ultra-deep sequencing approaches provide increased sensitivity for detecting minority variants. However, a phylogenetic a posteriori analysis revealed that most of the next-generation sequencing data published to date suffers from shortcomings. Because implementation of new technologies in clinical, population, or forensic studies requires proper verification, in this paper we present a direct comparison of ultra-deep 454 and Sanger sequencing for the detection of heteroplasmy in complete mitochondrial genomes of normal colon cells. The spectrum of heteroplasmic mutations is discussed against the background of mitochondrial DNA variability in human populations.

Knowledge of symptoms and diagnostic possibilities of cancer diseases

Introduction The aim of the present study was to analyse patients’ knowledge in the field of neoplastic prophylaxis. Material and methods The research was carried out between 2007 and 2008 in the Provincial Hospital in Bydgoszcz (i.e. general surgery, gynaecology and obstetrics, urology, breast surgery and thoracic surgery). Altogether 300 patients (of whom 250 were hospitalized) as well as 50 healthy subjects forming the control group were invited to participate in the study. A proprietary questionnaire containing eight multiple choice and another twelve open-ended questions was used for the purpose of the study. Results Prostate and lung cancer patients were more aware of their diseases compared to the control group, but the differences were not significant (p = 0.85 and p = 0.53 respectively). In the field of screening the patients’ knowledge, it was significantly higher in breast cancer subjects (p = 0.0008) while there was no difference compared to the control group in the remaining groups of cancer patients (i.e. colorectal, prostate or uterus cancer). Those most aware of their condition were patients from small towns (below 50,000), while subjects living in villages were the least aware. Conclusions Patients showed the greatest amount of knowledge regarding breast cancer and the least amount regarding prostate cancer. Oncological awareness in cancer patients was found to be related to variables such as education, age and residence. No difference was found between patients and controls, comparing their knowledge of disease symptoms as well as screening possibilities.

Urinary 5-hydroxymethyluracil and 8-oxo-7,8-dihydroguanine as potential biomarkers in patients with colorectal cancer

Abstract Context: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer. Objectives: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 5-hydroxymethyluracil (5-hmUra). Materials and methods: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited. Results: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was <0.7. The combination of these modifications showed better diagnostic power (AUC = 0.778 for 8-oxoGua + 8-oxodG)/5hmUra ratio). Conclusion: Urinary DNA modifications may reflect the oxidative stress/chronic inflammation in colorectal cancer but diagnostic performance for early-detection is moderate.

Usefulness of CEA Concentration Measurement and Classic Colonoscopy in Follow-Up After Radical Treatment of Colorectal Cancer

UNLABELLED There is always a certain rate of recurrence after radical treatment for cancer and to get on it an early detection of disease set back is crucial. MATERIAL AND METHODS Medical data of patients operated on for primarily detected colorectal cancer in years 1993-2002 was retrospectively reviewed. Usefulness of follow-up means such as physical examination, or CEA and endoscopic surveillance was analyzed. All mentioned above were applied to scheduled follow-up (in 3, 6 and 12 month intervals following an operation and annually after that by the year 5). RESULTS Complete and reliable data was obtained from 340 out of 502 follow-up intended subjects (67.7%). Elevated CEA was the most frequent predictor of recurrence within non-symptomatic subjects meeting follow-up appointments (60%). The cancer set back diagnosed by means of either physical or endoscopic examinations was the case only in one out of five patients (20.75% and 18.87% respectively). Clinical onset of recurrence making patients meet an unscheduled appointment was found increasing relative risk of nothing-but-palliative option either for them with local set back, or meta-static spread. Relative risk of onset of meta-chronous colonic cancer was significantly higher in patients being affected by synchronous advanced adenoma at time of surgery compared to those with one-fold changes. CONCLUSIONS CEA scheduled follow-up after treatment for colorectal cancer CRC seems adequate to provide a good outcome of treatment for recurrent tumors. CRC patients presenting with synchronous advanced adenomas at time of surgery are probably to be under more intensive endoscopic surveillance.