Discordant expression of hormone receptors and HER2 in breast cancer. A retrospective comparison of primary tumors with paired metachronous recurrences or metastases.

PURPOSE: Expression of biomarkers in breast cancer, such as the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), can impact therapeutic decisions; however, it has been reported that their expression may change with disease progression. The aim of this retrospective study was to investigate the expression of these biomarkers in primary breast cancer and in its metachronous recurrences or metastases, and to estimate the percentage of cases with discordant expression. METHODS: Paired primary and metastatic tumor samples were collected from patients with primary breast cancer and subsequent metachronous distant metastases, diagnosed at the Metaxa Cancer Hospital, Piraeus, Greece, from 1988 to 2008. Two cases of local recurrence were also included. ER, PR and HER2 expression were assessed by immunohistochemistry (IHC) according to ASCO-CAP 2007 guidelines. Statistical comparisons were made using McNemar's exact test and Bowker's test for symmetry. RESULTS: Tumor samples from 110 patients were analysed. In the primary tumor, ER, PR and HER2 were positively expressed in 64.5%, 58.2% and 32.7% of cases, respectively, and expression of these biomarkers was lost in 18.2%, 21.8% and 10.9% of the corresponding metastases, respectively. Overall, a change of ER, PR and HER2 expression from positive to negative and vice versa occurred in 27.3% (p = 0.0987), 25.5% (p < 0.001) and 18.2% (p = 0.5034) of the cases, respectively. CONCLUSION: The expression of ER, PR and HER2 in metachronous recurrences or metastases can be discordant from that observed in the primary tumor. As such changes can occur during disease progression, the evaluation of biomarkers in metastatic sites should be mandatory, whenever possible, to ensure that patients are receiving the most effective treatment at all times.

Triple-negative phenotype is of adverse prognostic value in patients treated with dose-dense sequential adjuvant chemotherapy: a translational research analysis in the context of a Hellenic Cooperative Oncology Group (HeCOG) randomized phase III trial.

PURPOSE: It is well recognized that breast cancer is a heterogeneous disease. The purpose of the current study was to classify patients according to the immunohistochemical phenotype of their tumors in an effort to evaluate the outcome of the respective groups of patients and specifically of those with triple-negative breast cancer (TNBC) following dose-dense sequential adjuvant chemotherapy. METHODS: A total of 595 patients with high-risk breast cancer were treated with adjuvant anthracycline-based dose-dense sequential chemotherapy with or without paclitaxel in the context of a randomized study. ER, PgR, HER2, Ki67, EGFR, and CK5 protein expression were evaluated in 298 formalin-fixed paraffin-embedded tumor samples by immunohistochemistry (IHC). HER2 was also evaluated by chromogen in situ hybridization (CISH). HER2 status and Ki67 protein expression differentiated luminal IHC subtypes (luminal B tumors being HER2 and/or Ki67-positive). RESULTS: Among the 298 tumors, the immunohistochemical panel classified 37 (12%) as luminal A, 198 (66%) as luminal B, 27 (9%) as HER2 enriched, and 36 (12%) as TNBC. The median follow-up time was 97 months. Patients with luminal A tumors had the best prognosis, with improved disease-free survival (log-rank, P = 0.033) and overall survival (P = 0.006) compared with the other three tumor subtypes. The three subtypes had an increased risk for relapse and death compared with luminal A in multivariate analysis, as well. No benefit from paclitaxel treatment was detected in any of the four subtypes or the total cohort. Hierarchical clustering based on mRNA expression of ER, PgR, and HER2 by quantitative RT-PCR identified patient groups that were comparable to the subtypes identified by IHC. CONCLUSIONS: The results of this study confirm that triple negative, luminal B and HER2-enriched phenotypes identified by IHC are of adverse prognostic value in high-risk breast cancer patients treated with dose-dense sequential adjuvant chemotherapy.

Analysis of 7 immunohistochemical markers in male germ cell tumors demonstrates the prognostic significance of p53 and MIB-1.

BACKGROUND: Various prognostic factors have been investigated in order to predict the minority of male germ cell tumor (GCT) patients who will develop resistant disease. However, no prognostic system has been proven accurate. MATERIALS AND METHODS: Paraffin-embedded tissue specimens, obtained from primary lesions during the initial diagnosis of 83 advanced chemotherapy-treated GCT male patients, were stained for 7 immunohistochemical markers: p53, bax, bcl-2, MIB-1, topoisomerase IIa, c-kit and COX-2. The percentage of positive cells for each marker was measured for each patient. Cox regression was used for the prognostic factor analysis. RESULTS: All patients were followed for a median of 4 years. Nineteen patients had seminoma and 64 non-seminomatous GCT. In univariate analysis, only p53 (hazard ratio (HR) = 4.01, 95% confidence interval (CI) = 1.25-12.84, p = 0.019) and MIB-1 (HR = 3.16, 95% CI = 1.06-9.45, p = 0.039) were found to be prognostic for disease-specific survival. The best prognostic cut-off values of p53 and MIB-1 were 10% and 30% respectively. In multivariate analysis, these two markers obtained independent significance only when considered in combination (HR = 6.63, 95% CI = 1.40-31.41, p = 0.017, for patients with one or both markers above their cut-off), while the International Germ Cell Consensus Cancer Group (IGCCCG) risk was the most significant (HR = 7.99, 95% CI = 1.96-32.52, p = 0.004, for the high-risk group). However, the expression of these markers seemed to be significantly correlated with known prognostic factors. Nevertheless, we identified 34 patients of low IGCCCG risk expressing both markers below their cut-off with excellent survival. CONCLUSION: Among 7 immunohistochemical markers, p53 and MIB-1 demonstrated prognostic significance. Their combination may contribute to improvement of the accuracy of the currently approved prognostic system (IGCCCG).

Complete response after imatinib mesylate administration in a patient with chemoresistant stage IV seminoma.

The case of a young man with stage IV chemoresistant pure seminoma overexpressing KIT, who achieved complete remission (CR) after the administration of imatinib mesylate (400 mg once daily), along with a third-line chemotherapy regimen, consisting of paclitaxel (150 mg/m2), oxaliplatin (100 mg/m2) and gemcitabine (800 mg/m2) every 2 weeks with granulocyte colony-stimulating factor (G-CSF) support is reported. The patient had received first- and second-line regimens consisting of ifosfamide, bleomycin, etoposide cisplatin (5 cycles, every 3 weeks) and methotrexate, vinblastine, actinomycin D, cyclophosphamide, cisplatin (3 cycles, every 3 weeks) respectively, without having normalized beta-human chorionic gonadotrophin (beta-HCG) levels. Following treatment with imatinib plus third-line chemotherapy (paclitaxel, oxaliplatin, gemcitabine), the levels of beta-HCG were reduced to within the normal limits during the first month of treatment. Therefore, the patient underwent surgical resection of the residual disease from the retroperitoneum and liver, which proved to be only necrotic tissue. The patient is under close follow-up, with no evidence of disease, 36 months after the completion of chemotherapy and 32 months post surgery.

Kit expression in male germ cell tumors.

OBJECTIVE: KIT functions as the receptor for stem cell factor (SCF) and this interaction is essential for regulation of proliferation and survival, particularly for germ cells since it regulates oogenesis, folliculogenesis and spermatogenesis. Up-regulation of KIT signalling has been associated with oncogenic transformation in cells expressing the molecule. Our objective was to investigate the expression of KIT in germ cell tumor patients and correlate it with the patients' clinical characteristics. PATIENTS AND METHODS: One hundred and seventy-three archival blocks of formalin fixed, paraffin-embedded tumor samples from histologically confirmed germ cell tumor (GCT) patients were included in the study. Immunohistochemical staining for KIT was performed and the percentage of positive cells was calculated by an independent pathologist. KIT expression was considered as positive if > 10% of tumor cells displayed membranous or cytoplasmic staining. RESULTS: Sixty-one patients with seminomatous (49 pure, 11 anaplastic, 1 spermocytic) and 112 with non-seminomatous GCTs (36 malignant teratoma undifferentiated (MTU), 15 malignant teratoma trophoblastic (MTT), 20 malignant teratoma intermediate (MTI), 35 malignant teratoma combined (MTC) and six others) were identified. Among pure seminoma patients, 38 (77.5%) revealed a positive staining for KIT, while only two out of eleven (18.2%) anaplastic seminoma patients were identified as positive. This difference was statistically significant (p < 0.001). Among 35 patients with an MTC, 48.6% had a positive KIT staining while only two of the remaining patients with a non-seminomatous GCT had a positive staining. Although KIT was strongly correlated with seminomatous histology (p < 0.001), it failed to correlate with stage (p = 0.19) or treatment response (p = 0.11) in these patients. Overall, four (one seminoma, three MTC) out of 22 chemoresistant patients showed a positive staining for KIT. CONCLUSION: KIT is expressed in the majority of seminomas and in seminomatous components of the combined tumors, but only in a minority of anaplastic seminomas and rarely in non-seminomatous GCTs. The recent development of tyrosine kinase inhibitors may offer a possibility of cure in chemoresistant patients overexpressing KIT.

HER-2/neu status of primary breast cancer and corresponding metastatic sites in patients with advanced breast cancer treated with trastuzumab-based therapy.

BACKGROUND: The aim of this prospective study was to investigate whether there were changes in HER-2/neu status in newly-developed metastatic lesions following treatment with trastuzumab in advanced breast cancer patients overexpressing HER-2/neu. The utility of serological assays for HER-2/neu in such patients was also studied. PATIENTS AND METHODS: Sixteen patients with HER-2/neu-overexpressing tumors (15 were 3+ by immunohistochemistry (IHC) and one 2+ by IHC and positive by the chromogenic in situ hybridization (CISH) test) were included in the study. Fourteen patients underwent biopsy and 2 patients fine-needle aspiration (FNA) of newly-developed metastatic lesions following trastuzumab treatment. All samples were assayed for HER-2 by IHC and by the CISH test. Serial serum HER-2/neu (S-HER-2) levels were measured prior to (baseline values) and during trastuzumab-based treatment by enzyme-linked immunosorbent assay (ELISA) (cut-off point: 10 ng/ml) in all patients. The patients were divided into 2 groups: those with "altered HER-2/neu status" and those with "conserved HER-2/neu status" in the metastatic region. RESULTS: Six out of the 16 (37%) ("altered HER-2/neu status") newly-developed metastatic lesions lost their HER-2/neu overexpression and scored 0 or +1 by IHC or negative on the CISH test, while in the remaining cases (10/16, 62.5%) ("conserved HER-2/neu status"), the HER-2/neu status was unchanged (+3 by IHC or a positive CISH test). Baseline S-HER-2 levels were elevated in 5 out of 16 patients (3 of "altered HER-2/neu status", 2 of "conserved HER-2/neu status"). The serum HER-2 (S-HER-2) levels declined and returned within the normal ranges in all these 5 patients as a response to trastuzumab treatment. Following the disease progression, the S-HER-2 levels of the 3 patients with "altered HER-2/neu status" remained normal, while those of 2 with "conserved HER-2/neu status" increased. There was no statistically significant difference in the number of chemotherapeutic treatments or the median time of treatment with trastuzumab or chemotherapy between the 2 groups. Time to tumor progression (TTP) was significantly shorter in the "altered HER-2/neu status" patients (median TTP for "altered HER-2/neu status": 9.5 months, and for "conserved HER-2/neu status": 12 months; p <0.001). CONCLUSION: These data suggest that, for most patients with metastatic breast cancer treated with trastuzumab, the HER-2/neu expression as measured by IHC and/or CISH in newly-developed metastatic lesions was unchanged. However, a remarkable percentage of cases lost HER-2/neu overexpression. It is not clear whether this finding implies resistance or sensitivity to trastuzumab.

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

DNA damage and mutagenesis induced by procarbazine in lambda lacZ transgenic mice: evidence that bone marrow mutations do not arise primarily through miscoding by O6-methylguanine.

The DNA damaging and mutagenic activities of procarbazine, a methylating drug employed in cancer chemotherapy and suspected of causing therapy-related leukaemia, were investigated in the liver and bone marrow of lambda lacZ transgenic mice (MutaMouse). The drug was administered using two different protocols, a 'high-dose' one involving 5 daily doses of 200 mg/kg, expected to cause depletion of the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) and thus favour the selective accumulation of the premutagenic lesion O6-methylguanine (O6-meG) relative to other adducts, and a 'low-dose' one involving 10 daily doses of 20 mg/kg procarbazine. Substantial accumulation of O6-meG was observed in both tissues examined 6 h after the end of the 'high-dose' treatment, with the liver accumulating somewhat higher levels than the bone marrow (28.0 +/- 1.8 fmol/microg DNA and 18.5 +/- 1.1 fmol/microg DNA respectively). However, significant increases in mutant frequency 10 days after the end of treatment were observed only in the bone marrow, reaching a 16-fold increase over background following the 5 x 200 mg/kg treatment. Sequence analysis of the mutations induced after this treatment revealed a mixed spectrum, in which G:C-->A:T transitions (characteristic of O6-meG miscoding) were only a secondary feature: Among 20 mutants analysed, only six such mutations were found, including three at CpG sites, which might have arisen from deamination of 5-methylcytosine. The other mutations observed included 1 A:T-->G:C transition, five transversions (one G:C-->T:A, one double G:C-->C:G, two A:T-->T:A, one A:T-->C:G), five deletions and three insertions. The mechanistic and clinical significance of these findings is discussed.

Prognostic significance of apoptosis related proteins Bcl-2 and Bax in node-negative breast cancer patients.

In this immunohistochemical study we investigated the expression of Bcl-2 and Bax apoptosis related proteins in node-negative breast carcinomas. The results were correlated with the recurrence rate of the patients. In order to avoid the influence of the most important tumor prognostic parameters we selected two groups of node negative breast ductal carcinomas that were grade II according to Bloom and Richardson classification, had a diameter of 1-3 cm but differed in clinical outcome: 44 of the patients had a 10 year disease-free survival while 46 developed metastatic disease in the same period of time. Bcl-2 and to a lesser degree Bax expression were inversely related with distant metastases (Pbcl-2 = 0.007, Pbax = 0.03). Combined analysis of Bax/Bcl-2 expression in relation to clinical outcome showed that the absence of both factors was more strongly associated with the development of distant metastases (P = 0.006, Ptrend = 0.001). Our findings indicate that Bcl-2 and Bax apoptosis-related proteins are good indicators of prognosis in node-negative breast cancer patients, and their combined absence, suggestive of serious deregulation of the apoptotic process, may contribute to the biologic aggressiveness of the tumors.

K-ras mutation in Greek patients with poorly and moderately differenciated tumours of the lower intestinal tract.

The K-ras protooncogene is activated via mutations in approximately 40% of primary colorectal adenocarcinoma. This finding suggests that these genetic alterations are important events in the genesis of colon cancer and should be correlated with other parameters in order to infer some conclusions relevant to the etiology and the pathogenesis of this type of cancer. In our study we examined whether the incidence of K-ras mutations detected in 23 samples with colorectal adenocarcinomas, was related to different anatomical sites within the lower intestinal tract (transverse colon, descending colon and rectosigmoid region) and also the correlation between K-ras mutations and depth of invasion, level of tumour cell differentiation and metastasis to the regional lymph nodes. For this study the critical regions for the activation of the K-ras protooncogene were amplified by the PCR technique and the particular sequences analysed, after their membrane transfer, by differential hybridization with selected synthetic oligonucleotides. Our results demonstrated that 39% of the adenocarcinomas examined contained point mutations, and 66.6% of these were located in the second position of K-ras codon 12, whereas the other 33.3% were located in the second position of codon 13. 77.8% of the mutations were located at the rectosigmoid region and the relevance of the mutations was higher in poorly differenciated tumours. The depth of invasion was associated with the presence of a mutation whereas no correlation was found between the presence of a mutation anb the regional lymph node metastasis.

DNA adducts and the mechanism of carcinogenesis and cytotoxicity of methylating agents of environmental and clinical significance.

DNA adducts are covalent complexes formed between genotoxic carcinogens and DNA bases, and constitute a critical early intermediate on the pathway of chemical carcinogenesis. Their accumulation in different tissues reflects the amount of activated carcinogen reaching DNA, and can therefore serve as an index of the biologically relevant dose reaching the target tissues or cells. Methylating agents are of interest in view of their occurrence in the environment and their use as cytotoxic drugs in cancer chemotherapy. Current evidence indicates that O6-methylguanine plays a particularly important role in the mutagenic, carcinogenic, and cytotoxic activities of methylating agents. O6-Methylguanine is repaired efficiently by the enzyme O6-alkylguanine-DNA alkyltransferase (AGT). Lack of this enzyme results in excessive accumulation of O6-methylguanine and recent evidence suggests that significant quantitative effects on adduct accumulation may be linked to conditions of very low AGT levels. This would be important from the point of view of clinical practice, since modulation of AGT is under investigation as a means of enhancing the therapeutic efficacy of clinical agents acting via the production of O6-methylguanine and related adducts, such as, for example, procarbazine, dacarbazine, and some nitrosoureas. The measurement of O6-methylguanine in human DNA has been employed as a tool to investigate the role of environmental methylating agents in human carcinogenesis. While the nature and origin of the methylating agents responsible for these adducts is currently unknown, recent studies in patas monkeys have shown that N-nitrosodimethylamine, a methylating carcinogen to which human exposure is well documented, is capable of efficiently generating O6-methylguanine in most tissues, including fetal tissues. Furthermore, it has been found that this damage is substantially enhanced by the coadministration of ethyl alcohol which acts by inhibiting the liver first-pass metabolism of the carcinogen, an observation which supports the hypothesis that alcohol consumption may act as a risk factor in human carcinogenesis by augmenting the action of nitrosamines.

Comparative study of the formation and repair of O6-methylguanine in humans and rodents treated with dacarbazine.

The mutagenic, carcinogenic and cytotoxic activity of dacarbazine, a drug employed in cancer chemotherapy, may be related to the induction in DNA of O6-methylguanine (O6-meG), a quantitatively minor but biologically important lesion. In the present study the kinetics of O6-meG formation and repair in blood leukocyte DNA were examined in 20 Hodgkins lymphoma patients treated i.v. with 180 +/- 13 (mean +/- SD) mg/m2 dacarbazine and compared with those observed in various tissues of rodents treated with different doses of the drug. In Hodgkin's lymphoma patients adduct levels reached a value of 0.27 +/- 0.14 fmol/microgram DNA 2 h after dacarbazine administration, while the rate of subsequent loss suggested an adduct half-life of < or = 30 h. Measurement of adduct levels in the same individuals after successive courses of treatment spaced 3 weeks apart (up to 10 treatment courses) demonstrated a consistent individual response and statistical analysis of variance confirmed that intra-individual variation in adduct accumulation after a given dose of dacarbazine accounted for only 5% of the total variance observed. In contrast, inter-individual variation accounted for 70% of the observed variance, with adduct levels 2 h after drug treatment varying approximately 7.5-fold among adduct-positive individuals. No significant depletion of lymphocyte O6-alkylguanine-DNA alkyltransferase (AGT) occurred after patient treatment with dacarbazine. No significant relationship between adduct levels and clinical response to treatment was observed. In rats treated with single or multiple doses of dacarbazine causing varying degrees of AGT depletion the highest levels of O6-meG were seen in the liver, followed by the lymph nodes, bone marrow and blood leukocytes, which showed up to approximately 2-fold lower levels. A similar tissue distribution was also observed in mice and in a single rabbit. These observations suggest that O6-meG levels assayed in blood leukocytes of therapeutically treated humans reflect those present in the -lymph nodes (target tissue for chemotherapy) and the bone marrow (target tissue for leukaemogenesis) and may be utilized as a measure of the drug dose reaching these tissues. The quantitative data reported in this study show that under conditions of no depletion of AGT O6-meG accumulates in blood leukocyte DNA of humans at a rate similar to that observed in rats, suggesting that human susceptibility to any O6-meG-mediated genotoxic effects of dacarbazine may be comparable with that of the rat.

Comparative dosimetry of O6-methylguanine in humans and rodents treated with procarbazine.

The accumulation of O6-methylguanine (O6-meG) in the DNA of blood leukocytes of 21 Hodgkin's lymphoma patients (followed for up to 12 cycles of treatment) treated in the context of MOPP combination chemotherapy with 150 mg procarbazine daily for 10 days was examined and compared to that observed in rats treated with different doses of procarbazine as a single agent once per day for 10 days. In humans, the adduct accumulated in a dose-related fashion and appeared to approach a steady-state after 7-8 days of treatment. Adduct levels on day 10 of the treatment cycle averaged 0.25 +/- 0.09 (mean +/- SD) mumol/molG and, for different individuals, covered a 3-fold range. Intra-individual variability between different treatment cycles was much more limited than inter-individual variability, the two parameters accounting for 8.9% and 84.5% respectively of adduct variance at a constant cumulative dose. Comparison of the dose-response relationships for humans and rats indicates that, under conditions of no depletion of O6-alkylguanine-DNA alkyltransferase (AGT), O6-meG accumulates in blood leukocyte DNA of humans at a rate which is only approximately 2-fold lower than in rats, implying that, to the extent to which O6-meG contributes to the genotoxic activity of procarbazine, human susceptibility to it is likely to be comparable to that of the rat. This is likely to be true also of the bone marrow (the tissue of interest as a target tissue for leukaemogenesis), since the tissue distribution of O6-meG induced by low doses of procarbazine in rats, mice and rabbits indicated that blood leukocyte levels of this adduct closely reflect those in the bone marrow. Based on these results, it is estimated that by the end of a MOPP chemotherapy cycle O6-meG reaches levels of the order of 0.2-0.3 fmol/microgram DNA (0.3-0.5 mumol/molG) in human bone marrow (the target tissue of leukaemogenesis observed after such treatment).

Differential effects of procarbazine and methylnitrosourea on the accumulation of O6-methylguanine and the depletion and recovery of O6-alkylguanine-DNA alkyltransferase in rat tissues.

The kinetics of accumulation of the premutagenic DNA adduct O6-methylguanine (O6-meG) in the liver, blood leukocytes, lymph nodes and bone marrow of rats was examined and compared after single or multiple doses of procarbazine, a methylating cytostatic drug employed in the treatment of Hodgkin's lymphoma patients, and methylnitrosourea (MNU), an experimental methylating agent and carcinogen. Maximal O6-meG levels occurred 1-2 h after administration of single doses of procarbazine (10 mg/kg) or MNU (1 mg/kg), thereafter decreasing with half-lives of approximately 20-45 h, depending on the tissue. A relatively uniform tissue distribution was observed with both agents, with the liver generally showing highest adduct levels, followed by the lymph nodes, bone marrow and blood leukocytes which contained broadly similar amounts of O6-meG. During daily, oral administration to rats of procarbazine for 10 days at dose rates of 2.5, 5, 10 or 20 mg/kg/day (treatment analogous to that of the MOOP chemotherapy protocol for Hodgkin's lymphoma) followed by animal death on different days (in each case 24 h after the last treatment), a biphasic mode of O6-meG induction was observed: an initially steep build-up during the first 3-4 days was followed by a transient decline in the rate of accumulation, in turn followed by a second wave of accumulation and then a further slow-down. During the same treatment, liver O6-methylguanine-DNA alkyltransferase (AGT) declined in a dose-related manner. AGT recovery after the end of treatment was slow, taking nearly 20 days after the end of the high-dose treatment to return to control levels, despite the fact that all detectable adducts had been lost from DNA within 3 days after the end of treatment. A similar depletion and slow recovery of AGT in the liver, blood lymphocytes, bone marrow and lymph nodes was observed after treatment with a single dose of 100 mg/kg procarbazine. In contrast to these observations, O6-meG accumulated smoothly during a 10 day administration of MNU (1 or 10 mg/kg/day) to reach a steady-state within 5-6 days, while liver AGT was partially depleted after the high dose and recovered fully within 72 h of cessation of treatment. Similarly, a single dose of MNU (35 mg/kg) resulted in AGT depletion followed by rapid recovery in all four tissues examined. It is concluded that procarbazine (but not MNU) causes a decrease in cellular AGT concentrations by a mechanism additional to suicide repair of O6-meG.(ABSTRACT TRUNCATED AT 400 WORDS)

Accumulation of O6-methylguanine in human DNA after therapeutic exposure to methylating agents and its relationship with biological effects.

O6-Methylguanine has been measured in peripheral blood leukocytes of 14 patients during one or more cycles of treatment with procarbazine (daily treatment for 10 days) and in 12 patients during one or more cycles of treatment with dacarbazine (single dose per cycle). Adduct formation at levels up to about 0.4 fmole/microgram DNA was detected in all procarbazine- and all but one dacarbazine-treated patients at some point after treatment. O6-Methylguanine accumulated during procarbazine treatment in a dose-related manner (mean rate of accumulation 2.8 x 10(-4) fmole/microgram DNA per mg/m2 dose) and appeared to approach a plateau by the end of the cycle (above 600 mg/m2 cumulative dose). The average rate of O6-methylguanine formation 2 hr after dacarbazine treatment was 11 +/- 8 x 10(-4) fmole/microgram DNA per mg/m2 dose. Individuals examined on more than one treatment cycle with either drug showed broadly similar methylation responses. The rate of adduct accumulation showed a nonsignificant, negative correlation with the pretreatment lymphocyte levels of the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) in the case of procarbazine and no correlation in the case of dacarbazine. No consistent lymphocyte AGT depletion was noted as a result of treatment with either drug. No correlation between O6-methylguanine formation and hematological toxicity was observed. In eight patients showing full remission after treatment with dacarbazine, the value of O6-methylguanine (averaged over all the cycles) was 0.252 +/- 0.120 fmole/microgram DNA while in four patients showing partial or no response it was 0.087 +/- 0.110 fmole/microgram DNA (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)