Predicting the myelotoxicity of chemotherapy: the use of pretreatment O6-methylguanine-DNA methyltransferase determination in peripheral blood mononuclear cells.

To assess the value of pretreatment O6-methylguanine-DNA methyltransferase (MGMT) expression in peripheral blood mononuclear cells (PBMCs) in predicting haematological toxicity with O6-alkylating agent chemotherapy, we explored this relationship retrospectively in melanoma patients. Ninety-three patients treated with temozolomide or dacarbazine in four clinical trials were assessed, and a model of the interaction between MGMT expression and haematological toxicity was constructed. Nadir white-cell and platelet counts were related to, and hence could be predicted from, pretreatment MGMT. Leucopenia and thrombocytopenia were more prevalent amongst patients with low pretreatment MGMT, according to the highest grades of toxicity experienced and/or the dose intensity patients could sustain. Addition of interferon to chemotherapy or compression of the temozolomide schedule increased the toxicity. The model also predicts significant myelotoxicity where PBMC MGMT is inactivated, consistent with the experience in the clinic with lomeguatrib and O6-benzylguanine. Determination of MGMT in PBMC can identify patients at greatest risk of toxicity or who are suitable for dose intensification.

A methionine-free diet associated with nitrosourea treatment down-regulates methylguanine-DNA methyl transferase activity in patients with metastatic cancer.

BACKGROUND: Methionine (MET) depletion used in association with chemotherapy improves the therapeutic index in animal models. This potentiating effect may be due to tumor cell sensitization to chloroethylnitrosoureas through their MET dependency and the down-regulation of O6- methylguanine-DNA methyltransferase (MGMT). Our purpose was to evaluate the impact of the association of a dietary MET restriction with nitrosourea treatment on MGMT activity in peripheral blood mononuclear cells (PBMCs). PATIENTS AND METHODS: Six patients with metastatic cancer (melanoma and glioma) received 4 cycles of a MET-free diet with cystemustine (60 mg/m2). RESULTS: MGMT activity in PBMCs decreased by an average of 13% from 553+/-90 fnol/mg before the diet to 413+/-59 fmol/mg after the diet + chemotherapy period (p=0.029). The decrease of MGMT activity was not affected by the duration of the MET-free diet period but seems to be correlated to the plasma MET depletion induced by the MET-free diet.

Significance of the O6-methylguanine-DNA methyltransferase and glutathione S-transferase activity in the sera of patients with malignant and benign ovarian tumors.

OBJECTIVE: To demonstrate O6-methylguanine-DNA methyltransferase (MGMT) and glutathione S-transferase (GST) activities by analyzing the sera separately obtained from patients with malignant ovarian tumors, benign ovarian tumors, and healthy individuals. STUDY DESIGN: Fourty-nine patients with ovarian cancer, nine patients with benign tumors, and 22 healthy women were included in this study. Blood samples were obtained from all the subjects in the malignant-tumor, benign-tumor, and control groups. Patients with malignant tumors underwent second and third phlebotomies one week following the surgery and after the chemotherapy regimen, respectively. MGMT, GST, and protein levels were measured for each serum sample. GST activity of the samples was measured by the method of Habig et al. using l-chloro-2-4 dinitrobenzene (CDNB) as substrate. MGMT activity was measured by the transfer of radio labelled methyl groups from a prepared MG-DNA substrate to the enzyme fraction of serum. Protein concentration was measured by biuret method. RESULTS: Our work demonstrated that untreated patients with malignant ovarian tumors revealed significantly greater MGMT and GST activities in their sera than did both healthy individuals and patients with benign ovarian tumors, while no significant difference was found between the healthy group and the patients with benign ovarian tumors with respect to their sera MGMT and GST activities. GST activity following chemotherapy was significantly lower than the postoperative values preceding chemotherapy. A relationship between sera MGMT and GST activities, tumor histology and pathology was not found in this study. CONCLUSION: Our work suggests the fact that detection of sera MGMT and GST activities is important in diagnostic and therapeutic approaches during the course of ovarian cancer.

Evaluation of O6-methylguanine DNA methyltransferase activity in patients with gastric cancer.

O6-Methylguanine DNA methyltransferase (O6-MGMT) reverses DNA alkylation damage produced alkylating agents. O6-MGMT is also a major determinant of cellular resistance to adjuvant chemotherapy with alkylating drugs. O6-MGMT activity was measured in samples from patients with gastric cancer, including tumor, adjacent normal appearing mucosa, and peripheral blood leukocytes (PBL). O6-MGMT activity of PBL from healthy individuals was evaluated as control. There was no significant difference between controls and patients for O6-MGMT activity in PBL. O6-MGMT activity was significantly increased in tumor tissue. Tumor O6-MGMT activity was found to be independent from tumor subgroups and tumor grade. A positive correlation was determined between O6-MGMT activity in tumor and in circulating PBL. The results indicate that O6-MGMT, a defense protein against alkylating agent-mediated carcinogenesis, increased in gastric tumors. This may explain the low response rate to drug combinations, including chloroethylnitrosoureas, exhibited by patients with gastric cancer.

Circadian variation in O6-alkylguanine-DNA alkyltransferase activity in circulating blood mononuclear cells of healthy human subjects.

Cytotoxic agents such as chloroethylnitrosoureas (CENUs) mostly alkylate DNA on the O6-guanine position. This highly mutagenic lesion can be repaired by O6-alkylguanine-DNA alkyltransferase (AGT), which removes the alkyl group by accepting it to the cysteine residue of its active site. AGT activity displayed a circadian rhythm in mouse liver, coincident with that of CENU tolerability. We investigated whether AGT activity displayed a circadian rhythm in human circulating mononuclear cells (MNCs). The study was performed in 12 healthy volunteers aged 19 to 31 years. Circadian synchronization was verified with rest/activity cycle as assessed with wrist actigraphy and plasma cortisol and melatonin rhythms. Subjects were hospitalized for 24 hr and blood samples were obtained at 08:00, 12:00, 16:00, 20:00, 22:00, 00:00, 02:00, 04:00 and 08:00 overnight. MNCs were isolated on Ficoll immediately after blood sampling and frozen at -196 degrees C until AGT activity determination by HPLC. Mean AGT activity (+/- SEM) varied from 821 +/- 67 fmol/mg of total proteins at noon (trough), up to 1,055 +/- 80 fmol/mg at midnight (peak), i.e., by approximately 30%. A circadian rhythm was statistically validated with both analysis of variance (p < 0.009) and cosinor (p < 0.02) for AGT activity in MNCs (acrophase +/- SD at 00:30 +/- 210 min) as well as for MNC circulating count and for plasma cortisol and melatonin concentrations. Despite individual variations in the extent of AGT activity rhythm (more or less pronounced according to subject), AGT activity displayed a circadian rhythm in human MNCs of our healthy study group subjects. The results warrant to further investigate AGT rhythmicity both in circulating MNCs and in target tissues of cancer patients, as a prerequisite for clinical testing of chronotherapy with alkylating agents.

Sequential administration of temozolomide and fotemustine: depletion of O6-alkyl guanine-DNA transferase in blood lymphocytes and in tumours.

BACKGROUND: The DNA repair protein O6-alkylguanine-DNA alkyl transferase (AT) mediates resistance to chloroethylnitrosoureas. Agents depleting AT such as DTIC and its new analogue temozolomide (TMZ) can reverse resistance to chloroethylnitrosoureas. We report the results of a dose finding study of TMZ in association with fotemustine. PATIENTS AND METHODS: Twenty-four patients with metastatic melanoma or recurrent glioma were treated with escalating dose of oral or intravenous TMZ ranging from 300 to 700 mg/m2, divided over two days. Fotemustine 100 mg/m2 was given intravenously on day 2, 4 hours after TMZ. AT depletion was measured in peripheral blood mononuclear cells (PBMCs) and in selected cases in melanoma metastases and was compared to TMZ pharmacokinetics. RESULTS: The maximum tolerated dose (MTD) of TMZ was 400 mg/m2 (200 mg/m2/d) when associated with fotemustine the 2nd day with myelosuppression as dose limiting toxicity. The decrease of AT level in PBMCs was progressive and reached 34% of pretreatment values on day 2. There was however wide interindividual variability. AT reduction was neither dose nor route dependent and did not appear to be related to TMZ systemic exposure (AUC). In the same patients, AT depletion in tumour did not correlate with the decrease of AT observed in PBMCs. CONCLUSIONS: PBMCs may not be used as a surrogate of tumour for AT depletion. Further study should concentrate on the pharmacokinetic pharmacodynamic relationship in tumour to provide the basis for individually tailored therapy.

Activity of O6-methylguanine DNA methyltransferase in mononuclear blood cells of formaldehyde-exposed medical students.

A recent study reported that exposure of student embalmers in Cincinnati to high concentrations of formaldehyde (2 mg/m3) reduced the activity of the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). Reduction in a DNA repair enzyme may strongly increase the cancer risk not only with respect to the repair-enzyme causing agent but with respect to all carcinogens causing lesions subject to repair by the enzyme in question. Thus, we examined whether formaldehyde exposure of 57 medical students during their anatomy course at two different Universities in Germany influenced MGMT activity in mononuclear blood cells. Mean formaldehyde exposure of 41 students was 0.2 +/- 0.05 mg/m3 for 6 h per week. MGMT activity was 133.2 +/- 14.9 fmol MGMT/10(6) cells before the beginning of the formaldehyde exposure, 131.1 +/- 15.8 fmol MGMT/10(6) cells after 50 days (P = 0.56) and 128.2 +/- 19.0 fmol MGMT/10(6) cells after 111 days of exposure (P = 0.92). Similarly, no significant influence of formaldehyde exposure was observed, when smoking habits, alcohol consumption, allergic disease and sex of students were considered. In addition no significant difference was obtained in MGMT activity between 16 students with mean formaldehyde exposure of 0.8 +/- 0.6 mg/m3 and students without formaldehyde exposure (n = 51; P = 0.37). In conclusion, exposure of the medical students in western Europe to formaldehyde did not decrease MGMT activity in mononuclear blood cells.

O6-methylguanine-DNA methyltransferase in pretreatment tumour biopsies as a predictor of response to temozolomide in melanoma.

Resistance of tumour cells to methylating and monochloroethylating agents in vitro and in vivo has been linked to levels of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). In a clinical trial of temozolomide in advanced malignant melanoma, the relationship between pretreatment MGMT levels in biopsies of cutaneous tumours and involved lymph nodes and clinical response to the drug has been studied. Among 50 evaluable patients, there were three complete responses (CR), four partial responses (PR), six with stable disease (SD) and 37 with progressive disease (PD), with an overall response rate of 14%. In 33 patients in whom MGMT level and clinical response could be evaluated, the tumour MGMT levels (fmol mg(-1) protein) were: CR, 158 +/- 119; PR, 607 +/- 481; NC, 171 +/- 101; PD, 185 +/- 42.3. Thus, measurements of pretreatment levels of MGMT in melanoma did not predict for response to temozolomide.

Heterogeneity of O6-alkylguanine-DNA-alkyltransferase measured by flow cytometric analysis in blood and bone marrow mononuclear cells.

Alkyltransferase (AGT) repairs alkylation at O6-guanine in DNA and is a major determinant of susceptibility to alkylating chemotherapeutic agents and carcinogens. Using a newly developed flow cytometry assay with the monoclonal anti-AGT antibody, mT3.1, we compared AGT expression in single-cell suspensions with standard biochemical and Western blot assays to validate the fluorescence-activated cell sorting (FACS) method and develop potential applications. From Chinese hamster ovary cells (CHO) transfected with human O6-methylguanine-DNA methyl-transferase cDNA, 6 CHO-O6-methylguanine-DNA methyl-transferase clones were isolated that expressed 0.3 to 64 fmol/microgram DNA (by biochemical assay) of human AGT. FACS yielded a linear relationship between mean fluorescence intensity and both AGT activity by biochemical assay and AGT protein by Western blot. Using this standard curve, FACS-analyzed AGT protein content in human peripheral blood mononuclear cells (PBMCs) from normal donors ranged from 6.1 to 12.8 fmol/microgram DNA, similar to those obtained by biochemical assay and Western blot. This suggests that the level of immunoreactive protein appears to be an accurate predictor of AGT activity in the steady state. FACS-AGT in PBMCs from normal donors had a low index of heterogeneity within the sample. In contrast, by FACS-AGT analysis of human bone marrow samples and granulocyte-colony-stimulating factor-mobilized PBMCs, AGT was lower and had an 8-fold higher index of heterogeneity than observed in PBMCs from normal donors. After treatment with O6-benzylguanine (O6-bG), Western and FACS-AGT detected significant levels of AGT protein for up to 24 h, whereas biochemical assay showed AGT activity less than 5% of the basal level. Because only the biochemical assay accurately measures net AGT activity, the AGT-FACS assay will not be useful in clinical trials to assess the efficacy of O6-bG or other AGT inhibitors. Thus, AGT-FACS can rapidly assess the heterogeneity of steady-state AGT in single-cell suspensions and may be useful for assay in lymphocytes, bone marrow cells, leukemic myeloma plasma cells, or cells transfected with the AGT gene; Western blot analysis is better for small samples such as tumor biopsies, whereas biochemical assay is best able to measure enzyme activity and its inactivation by O6-bG or other agents.

A phase II study evaluating the effect of tamoxifen on DNA repair in melanoma patients treated with dacarbazine.

The addition of tamoxifen to dacarbazine containing chemotherapy regimens used in the treatment of melanoma, has been shown to increase response rates, but the mechanism of any interaction is uncertain. The object of this study was to determine whether the addition of tamoxifen to dacarbazine, would modify DNA repair in-vivo and cause an increase in O6-meG adducts in peripheral blood leucocytes. This would provide some insight into the nature of the interaction between these two drugs. Twenty three patients with metastatic malignant melanoma received dacarbazine (DTIC) 1 g/m2 every three weeks for a maximum of six cycles. Tamoxifen 20 mg daily, was started after the first cycle of chemotherapy and then taken continuously during the treatment. Adduct levels after the second cycle of treatment were significantly higher than those after the first cycle (p = 0.0001). A similar rise however, was also produced when a cohort of patients were given dacarbazine without tamoxifen during the second cycle of treatment. This study did not show an additional increase of O6-meG adducts when tamoxifen was administered and therefore this mechanism does not support a postulated interaction between tamoxifen and dacarbazine. This is in agreement with the recent randomised study which did not show any significant increase in response rate with the addition of tamoxifen.