Krzysztof Roszkowski

Severe oxidatively damaged DNA after cisplatin treatment of cancer patients.

There is growing evidence suggesting that cytotoxic activity of cisplatin is closely associated with increased generation of reactive oxygen species (ROS). Therefore, this study was undertaken to examine oxidative DNA damage, which arises as a result of chemotherapy with cisplatin. Using HPLC prepurification/isotope dilution GC/MS methodology, we examined the amount of 8‐oxoGua and 8‐oxodG excreted into urine in cancer patients (n = 66) who received chemotherapy with cisplatin. One day after the infusion of the drug, significant increase in the amount of 8‐oxoGua and 8‐oxodG in urine of the patients was observed, when compared to the initial value (78%, p < 0.0001 and 22%, p = 0.0051). In the “nadir days” (when the most distinct cell death based on hematological cell counts can be observed), the level of modified base and nucleoside decreased in comparison with the aforementioned time point. These results, for the first time, indicate that oxidatively damaged DNA may be, at least in part, responsible for cisplatin induced cytotoxicity. Our results also demonstrate that cell death does not contribute to urinary 8‐oxoGua and 8‐oxodG in humans.

Melanin content in melanoma metastases affects the outcome of radiotherapy.

Melanin possess radioprotective and scavenging properties, and its presence can affect the behavior of melanoma cells, its surrounding environment and susceptibility to the therapy, as showed in vitro experiments. To determine whether melanin presence in melanoma affects the efficiency of radiotherapy (RTH) we evaluated the survival time after RTH treatment in metastatic melanoma patients (n = 57). In another cohort of melanoma patients (n = 84), the relationship between melanin level and pT and pN status was determined. A significantly longer survival time was found in patients with amelanotic metastatic melanomas in comparison to the melanotic ones, who were treated with either RTH or chemotherapy (CHTH) and RTH. These differences were more significant in a group of melanoma patients treated only with RTH. A detailed analysis of primary melanomas revealed that melanin levels were significantly higher in melanoma cells invading reticular dermis than the papillary dermis. A significant reduction of melanin pigmentation in pT3 and pT4 melanomas in comparison to pT1 and T2 tumors was observed. However, melanin levels measured in pT3-pT4 melanomas developing metastases (pN1-3, pM1) were higher than in pN0 and pM0 cases. The presence of melanin in metastatic melanoma cells decreases the outcome of radiotherapy, and melanin synthesis is related to higher disease advancement. Based on our previous cell-based and clinical research and present research we also suggest that inhibition of melanogenesis can improve radiotherapy modalities. The mechanism of relationship between melanogenesis and efficacy of RTH requires additional studies, including larger melanoma patients population and orthotopic, imageable mouse models of metastatic melanoma.

Oxidative damage DNA: 8-oxoGua and 8-oxodG as molecular markers of cancer

Summary Background The broad spectrum of oxidative damage DNA biomarkers: urinary excretion of 8-oxodG (8-oxo-7,8-dihydro-2′-deoxyguanosine), 8-oxoGua (8-oxo-7,8-dihydroguanine) as well as the level of oxidative damage DNA in leukocytes, was analyzed in cancer patients and healthy subjects. Material/Methods 222 cancer patients and 134 healthy volunteers were included in the analysis, using methodologies which involve HPLC (high-performance liquid chromatography) prepurification followed by gas chromatography with isotope dilution mass spectrometry detection and HPLC/EC. Results For the whole patient population (n=222) the median values of 8-oxoGua and 8-oxodG in urine samples were 12.44 (interquartile range: 8.14–20.33) [nmol/24 hr] and 6.05 (3.12–15.38) [nmol/24 hr], respectively. The median values of 8-oxoGua and 8-oxodG in urine samples of the control group (n=85) were 7.7 (4.65–10.15) [nmol/24 hr] and 2.2 (1.7–2.8) [nmol/24 hr], respectively. The level of 8-oxodG in DNA isolated from leukocytes of the patient population (n=179) and of the control group (n=134) was 4.93 (3.46–9.27) per 10’6 dG and 4.46 (3.82–5.31) per 10’6 dG, respectively. Conclusions The results suggest that oxidative stress in cancer patients, demonstrated by augmented amounts of these modifications in urine, could be typical not only for affected tissue but also for other tissues and even the whole organism. An assay that enables the determination of levels of basic markers of oxidative stress might be applied in clinical practice as an additional, helpful marker to diagnose cancer.

Selenium Supplementation Reduced Oxidative DNA Damage in Adnexectomized BRCA1 Mutations Carriers

Some experimental evidence suggests that BRCA1 plays a role in repair of oxidative DNA damage. Selenium has anticancer properties that are linked with protection against oxidative stress. To assess whether supplementation of BRCA1 mutation carriers with selenium have a beneficial effect concerning oxidative stress/DNA damage in the present double-blinded placebo control study, we determined 8-oxodG level in cellular DNA and urinary excretion of 8-oxodG and 8-oxoGua in the mutation carriers. We found that 8-oxodG level in leukocytes DNA is significantly higher in BRCA1 mutation carriers. In the distinct subpopulation of BRCA1 mutation carriers without symptoms of cancer who underwent adnexectomy and were supplemented with selenium, the level of 8-oxodG in DNA decreased significantly in comparison with the subgroup without supplementation. Simultaneously in the same group, an increase of urinary 8-oxoGua, the product of base excision repair (hOGG1 glycosylase), was observed. Therefore, it is likely that the selenium supplementation of the patients is responsible for the increase of BER enzymes activities, which in turn may result in reduction of oxidative DNA damage. Importantly, in a double-blinded placebo control prospective study, it was shown that in the same patient groups, reduction in cancer incidents was observed. Altogether, these results suggest that BRCA1 deficiency contributes to 8-oxodG accumulation in cellular DNA, which in turn may be a factor responsible for cancer development in women with mutations, and that the risk to developed breast cancer in BRCA1 mutation carriers may be reduced in selenium-supplemented patients who underwent adnexectomy. (Cancer Epidemiol Biomarkers Prev 2009;18(11):2923–8)

Small field radiotherapy of head and neck cancer patients is responsible for oxidatively damaged DNA/oxidative stress on the level of a whole organism

It is possible that oxidatively damaged DNA which arises as a result of radiotherapy may be involved in the therapeutic effect of the ionizing radiation and in the side effects. Therefore, for the first time, the broad spectrum of oxidatively damaged DNA biomarkers: urinary excretion of 8‐oxodG (8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine), 8‐oxoGua (8‐oxo‐7,8‐dihydroguanine) as well as the level of oxidatively damaged DNA in leukocytes, was analyzed in head and neck cancer patients (n = 27) undergoing fractionated radiotherapy using methodologies which involve HPLC (high‐performance liquid chromatography) prepurification followed by gas chromatography with isotope dilution mass spectrometry detection and HPLC/EC. Of all the analyzed parameters in the majority of patients, only urinary excretion of the modified nucleoside significantly increased over the initial level in the samples collected 24 hr after the last fraction. However, for the distinct subpopulation of 10 patients, a significant increase in the level of 8‐oxodG in cellular DNA and a simultaneous drop in urinary 8‐oxoGua (the repair product of oxidative DNA damage) were detected after completion of the therapy. Because 8‐oxoGua is a repair product of the DNA damage, there is a possibility that, at least in the case of some patients with the lowest activity of OGG1 (8‐oxo‐7,8‐dihydroguanine glycosylase), the combination of lower OGG1 repair efficacy and irradiation was associated with increased background level of 8‐oxoGua in cellular DNA. Apparently reduced DNA repair is unable to cope with the radiation‐induced, and the extra amount of 8‐oxoGua leading to an increase of potentially mutagenic/carcinogenic lesions.

Urinary 8-Oxoguanine as a Predictor of Survival in Patients Undergoing Radiotherapy

Background: Because of the importance to identify prognostic indicator for radiotherapy, herein we decided to check whether the parameters which describe oxidative stress/DNA damage may be used as a marker of the therapy. The aim of this work was to investigate whether fractionated radiotherapy of patients with cancer (n = 99) is responsible for oxidative DNA damage on the level of the whole organism and whether the biomarkers of the damage such as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) and its modified base 8-oxo-7,8-dihydroguanine (8-oxo-Gua) in urine and DNA may be used as a predictor of radiotherapy success. Methods: All the aforementioned modifications were analyzed using techniques which involve high-performance liquid chromatography/electrochemical detection (HPLC/EC) or HPLC/gas chromatography-mass spectroscopy (GC-MS). Results: Of all analyzed parameters only patients with significantly elevated urinary excretion of the 8-oxo-Gua with concomitant unchanged level of 8-oxo-dG in leukocytes DNA in the samples collected 24 hours after the first fraction in comparison to the initial level have significantly increased survival time (60 months after the treatment, survival of 50% of the patients who fulfill the above mentioned criteria, in comparison with 10% of the patients who did not). Conclusions: Results of our work suggest that patients with higher urinary 8-oxo-Gua and concomitant stable level of 8-oxo-dG in leukocytes DNA, after 24 hours of the first dose should be regarded as better responder to radiotherapy as being at lower risk of mortality. Impact: The above mentioned statement could make it possible to use these parameters as markers to predict the clinical success. Cancer Epidemiol Biomarkers Prev; 21(4); 629–34. ©2012 AACR.

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.

Analysis of Treg cell population alterations in the peripheral blood of patients treated surgically for ovarian cancer - a preliminary report.

Citation Wicherek L, Jozwicki W, Windorbska W, Roszkowski K, Lukaszewska E, Wisniewski M, Brozyna AA, Basta P, Skret‐Magierlo J, Koper K, Rokita W, Dutsch‐Wicherek M. Analysis of Treg cell population alterations in the peripheral blood of patients treated surgically for ovarian cancer – a preliminary report. Am J Reprod Immunol 2011; 66: 444–450

Cancer treatment in pregnant women

In general, strategies for the treatment of cancer in pregnancy should not differ significantly from the treatment regimens in non-pregnant women. However, this is difficult due to either the effects of anticancer drugs on the developing foetus or the possibility of long-term complications after the exposure to drugs and radiation. The decision about the introduction and continuation of treatment in the event of pregnancy should be preceded by a detailed analysis of the potential benefits and risks. There are no data to suggest that pregnancy termination alters the biological behaviour of the tumour or patient prognosis in the presence of appropriate antineoplastic therapy. All patients should be given appropriate advice and informed that there are insufficient scientific data to determine any generally accepted consensus. It is very important to always respect the will of the patient, and the moral judgment of the physician should have no impact on the decisions taken by the woman. If the woman decides to undergo active treatment and maintain her pregnancy, it is necessary to carry out consultations with experts in the field appropriate to the type of cancer. This paper presents a basic review of the literature on the targeted therapies currently used in selected cancers diagnosed during pregnancy: breast cancer, cervical cancer, Hodgkins disease, melanoma, thyroid cancer, ovarian cancer, and colorectal cancer.

Potential Role of Methylation Marker in Glioma Supporting Clinical Decisions

The IDH1/2 gene mutations, ATRX loss/mutation, 1p/19q status, and MGMT promoter methylation are increasingly used as prognostic or predictive biomarkers of gliomas. However, the effect of their combination on radiation therapy outcome is discussable. Previously, we demonstrated that the IDH1 c.G395A; p.R132H mutation was associated with longer survival in grade II astrocytoma and GBM (Glioblastoma). Here we analyzed the MGMT promoter methylation status in patients with a known mutation status in codon 132 of IDH1, followed by clinical and genetic data analysis based on the two statuses. After a subtotal tumor resection, the patients were treated using IMRT (Intensity-Modulated Radiation Therapy) with 6 MeV photons. The total dose was: 54 Gy for astrocytoma II, 60 Gy for astrocytoma III, 60 Gy for glioblastoma, 2 Gy per day, with 24 h intervals, five days per week. The patients with MGMT promoter methylation and IDH1 somatic mutation (OS = 40 months) had a better prognosis than those with MGMT methylation alone (OS = 18 months). In patients with astrocytoma anaplasticum (n = 7) with the IDH1 p.R132H mutation and hypermethylated MGMT, the prognosis was particularly favorable (median OS = 47 months). In patients with astrocytoma II meeting the above criteria, the prognosis was also better than in those not meeting those criteria. The IDH1 mutation appears more relevant for the prognosis than MGMT methylation. The IDH1 p.R132H mutation combined with MGMT hypermethylation seems to be the most advantageous for treatment success. Patients not meeting those criteria may require more aggressive treatments.