Ifosfamide metabolism and DNA damage in tumour and peripheral blood lymphocytes of breast cancer patients

Purpose: This study was designed to determine individual variation in the metabolism of ifosfamide (IF) and any influence this may have on the degree of DNA damage produced in both peripheral blood lymphocytes (PBL) and in tumour tissue. Methods: The pharmacokinetics and metabolism of IF and also of doxorubicin (DOX) were determined in patients receiving IF/DOX neoadjuvant chemotherapy for the treatment of advanced breast cancer. The DNA-damaging effects of this regimen were measured using the comet assay in PBL and in breast tumour tissue obtained by fine needle aspirate. Parallel in vitro studies were carried out in order to establish if DNA damage caused by IF metabolites or DOX was predictive of cytotoxicity in breast cancer cell lines. Results: The median AUC, half-life and clearance of IF were found to be 291 μM · min, 5.2 h and 66 ml/min per m², respectively. A high degree of interpatient variability (up to sevenfold) was observed in the metabolism of both IF and DOX and also in their metabolites. Treatment-related changes in the amount of DNA damage were observed in both PBL and tumour cells. That in PBL peaked 48 h after the end of IF infusion (median 17% damaged cells at 48 h compared to 4% damaged before treatment). DNA damage in tumour cells was not elevated above low pretreatment values (median 1.5% damaged cells) until 3 weeks after IF and DOX treatment (median 30% damaged cells), by which time damage in PBL showed almost complete resolution to basal levels. The DNA damage in PBL determined 24 h after the start of chemotherapy was found to be related to the AUC of 4-hydroxyifosfamide (4OHI; P=0.05). The amount of damage in either tissue did not significantly correlate with clinical response or toxicity, but lower amounts of damage were observed in the tumour cells 3 weeks after treatment in those patients that subsequently relapsed, compared to those that remained disease free. DNA damage (more than 20% damaged cells) was observed after exposure to active IF metabolites at concentrations equal to or greater than the IC₅₀ in MCF-7 and MDA-MB231 cell lines. At concentrations of 4OHI similar to those determined in vivo, an equivalent level of DNA damage was observed in PBL and in cell lines and was associated with significant growth inhibition. DNA damage induced by DOX was not predictive of cytotoxicity. Conclusion: Systemic DNA damage appeared to be related to levels of the active metabolite, consistent with the results of in vitro investigations of DNA damage. Further studies are warranted to substantiate this observation and to explore the relationship between metabolism, DNA damage and antitumour activity. Received: 11 February 2000 / Accepted: 20 July 2000     

Induction of DNA damage, inhibition of DNA synthesis and suppression of c-myc expression by the anthracycline analog, idarubicin (4-demethoxy-daunorubicin) in the MCF-7 breast tumor cell line

Purpose: Studies were designed to elucidate the basis for the antiproliferative activity of the anthracycline antibiotic, idarubicin (4-demethoxy-daunorubicin) in MCF-7 breast tumor cells. Methods: Growth inhibition was evaluated using the MTT tetrazolium dye assay, induction of DNA strand breaks was determined by alkaline elution, inhibition of DNA synthesis was assessed by measuring the incorporation of labelled thymidine into DNA, modulation of the expression of the c-myc oncogene was determined by Northern blotting and the induction of apoptosis was evaluated by alkaline unwinding, static field gel electrophoresis, terminal end labelling and assessment of cell morphology. Results: MCF-7 cells were relatively sensitive to idarubicin, with an IC ₅₀ value for growth inhibition of approximately 0.01 μM. While DNA strand breakage was not evident below a concentration of 0.1 μM idarubicin, where growth inhibition exceeded 70%, both the inhibition of DNA synthesis and suppression of c-myc expression closely paralleled the profile of antiproliferative activity for idarubicin. Finally, while exposure to idarubicin resulted in a substantial loss of viable cells within 48–72 h, there was no morphological evidence of apoptotic body formation. The absence of apoptosis in cells exposed to idarubicin was supported by studies demonstrating the absence of DNA fragmentation using gel electrophoresis, alkaline elution and in situ DNA end-labelling assays. Conclusions: The results of these studies extend previous results from this laboratory indicating an association between suppression of c-myc expression, inhibition of DNA synthesis and growth arrest by topoisomerase II inhibitors, as well as the lack of induction of apoptotic cell death by topoisomerase II inhibitors in MCF-7 breast tumor cells. Received: 21 May 1997 / Accepted: 10 September 1997     

Tumour Topoisomerase II Alpha Protein Expression and Outcome After Adjuvant Dose-Dense Anthracycline-Based Chemotherapy

There is a need for the selection of those breast cancers where benefit may be attained from the addition of an anthracycline to the adjuvant chemotherapy. The expression of topoisomerase II alpha (TOP2A) protein in 3 cohorts of breast cancers treated with adjuvant dose-dense anthracycline-based chemotherapy was determined retrospectively. The TOP2A status was analysed in relation with the other standard tumour features and the outcome. TOP2A IHC results were assessable in 106 patients: with a cut-off value of 15%, 48% of the tumours were classified as TOP2A-positive. The expression of TOP2A correlated with that of Ki67 (R = 0.532, p < 0.001) and a high grade (p = 0.04), but did not correlate with the proportion of ER- or PR-positive cells in the tumour. More tumors were TOP2A-negative among the ER- or PR-positive cancers than among the ER/PR-negative cancers (p = 0.021 and p = 0.002, respectively). After a median follow-up time of 64.5 months, 31 relapses (23.5%) and 23 deaths (17.4%) had occurred in 131 patients. The overall survival was longer in the TOP2A-positive cases than in the TOP2A-negative cases. The recurrence-free survival and the overall survival were significantly more favourable in the ER/PR-negative and TOP2A-positive tumours than in other subgroups. In a Cox proportional hazards model, the grade and TOP2A remained significant determinants in the ER/PR-negative subgroup. TOP2A positivity and grade 3 indicated a decrease in the risk of death with HR = 0.211 (95% CI: 0.042–1.05, p = 0.056) and HR = 0.216 (95% CI: 0.047–0.990, p = 0.048), respectively. A higher sensitivity to anthracycline-containing regimens is suggested in ER/PR-negative and TOP2A-positive cancers.     

Induction chemotherapy response and recurrence rates in correlation with N0 or N+ stage in oral squamous cell cancer (OSCC)

The authors compared N0 with N+ cases in neoadjuvant chemotherapy regression and recurrence. During a 12-year period, 180 consecutive oral squamous cell cancer patients were observed. Of these patients, 78 were N0 and 102 N+ stages. The drugs used were as follows: bleomycin, vincristine, methotrexate, and mitolactol. After three courses of chemotherapy, the regression (complete response (CR), partial response (PR), and no response (NR)) and side effect rate were determined. All patients were operated on and observed for the number and localization of recurrences during 3 year follow-up time. The N0 cases came from T2–3, while N+ was from T2–4a (AJCC 2002). The regression in the N0 group was CR 46%, PR 53%, and NR 1%; but in the N+ group, it was CR 12%, PR 72%, and NR 16%. The regression rate was significantly higher (p = 0.00025) for N0 group than N+ group. The regression rate for T3 N0 was significantly higher (p = 0.055) than for T3 N+ cases. In the N0 stage, the regression rate was significantly higher (p = 0.0174) for T2 than T3. In N+ stage, there was no significant difference (p = 0.183) for T2–4a. The side effects were slight. The recurrence rate for the N0 group was significantly lower (15%, p = 0.000069), while for N+ group, it was 59%. The dependence in the T3 cases was also significant (p = 0.009) in the 3-year tumor-free survival. The N stage seems a more important prognostic factor for chemotherapy response and recurrence rate than the T stage. Stage III can be divided into subgroups without metastasis (III.a) and with metastasis (III.b.), based on significant difference in regression and recurrence rate.     

Combination of nedaplatin and vindesine for treatment of relapsed or refractory non-small-cell lung cancer

Purpose: A phase II study of nedaplatin and vindesine was conducted to evaluate their efficacy and safety for treatment of relapsed or refractory non-small-cell lung cancer (NSCLC). Methods : Between August 1996 and September 1998, 48 patients who had previously received chemotherapy, thoracic radiotherapy, and/or surgery were enrolled in the study. Patients were required to have an Eastern Cooperative Oncology Group performance status of 0 to 2 and an age between 20 and 79 years. Treatment consisted of nedaplatin (80 mg/m², day 1) and vindesine (3 mg/m², days 1 and 8) every 3 to 4 weeks. Results: Of 48 patients, 7 (14.6%) exhibited an objective response. Four (50%) of eight chemotherapy-naive patients had a partial response. However, of the 40 patients who had received prior chemotherapy, a partial response was observed in only 3 (7.5%). At a median follow-up time of 85.1 weeks, the median survival time was 43.6 weeks (95% confidence interval 34.4–52.7) for patients who had received chemotherapy, with a survival rate of 40% at 1 year. Grade 3 or 4 neutropenia occurred in 43 of 48 patients (90%), and neutropenic fever was observed in 3 of the 43 patients, one of whom died of sepsis. Pharmacokinetic and pharmacodynamic analyses of platinum were performed in 43 patients during the first cycle of chemotherapy. Percent reduction in absolute neutrophil count was correlated not only with the area under the plasma ultrafilterable platinum concentration versus time curve (r=0.41, P=0.007) but also with the duration of ultrafilterable platinum concentration above 1 μg/ml (r=0.41, P=0.007). Patients with progressive disease exhibited a shorter duration of ultrafilterable platinum concentration over 1 μg/ml (P=0.046) than those with other responses. Conclusion: A combination of nedaplatin and vindesine was unsatisfactory as second-line chemotherapy for NSCLC, although the combination was well tolerated. The duration of ultrafilterable platinum concentration above 1 μg/ml was an important pharmacokinetic parameter for predicting both chemotherapy-induced neutropenia and treatment outcome. Received: 4 November 1999 / Accepted: 28 April 2000     

Genetic polymorphisms of ALDH2 and ADH2 are not associated with risk of hepatocellular carcinoma among East Asians

The aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 2 (ADH2) genes have been implicated in the development of hepatocellular carcinoma (HCC). However, the results have been inconsistent. In this study, we performed a meta-analysis to clarify the associations between polymorphisms of ALDH2 and ADH2 genes and HCC. Published literatures from PubMed and Embase were retrieved. Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated using fixed- or random-effects model. Ten studies including 1,231 HCC cases and 1,849 controls were included in the meta-analysis of the association between ALDH2 polymorphism and HCC risk. The results indicated that ALDH2 polymorphism was not significantly associated with risk of HCC (homogeneous co-dominant model: OR = 0.99, 95% CI 0.72–1.34; heterogeneous co-dominant model: OR = 0.90, 95% CI 0.75–1.08; dominant model: OR = 0.91, 95% CI 0.70–1.18; recessive model: OR = 1.11, 95% CI 0.66–1.87). In addition, four studies including 518 cases and 607 controls were included in the meta-analysis of the association between ADH2 polymorphism and HCC risk. There was no association between ADH2 polymorphism and HCC risk (homogeneous co-dominant model: OR = 0.93, 95% CI 0.58–1.51; heterogeneous co-dominant model: OR = 1.39, 95% CI 0.87–2.23; dominant model: OR = 1.19, 95% CI 0.76–1.88; recessive model: OR = 0.91, 95% CI 0.54–1.54). Further analysis suggested that the ALDH2 polymorphism–alcohol interaction was marginally associated with HCC risk under the dominant model (OR = 2.05, 95% CI 1.01–4.17). However, the result was not robust by sensitivity analysis. The results from the present meta-analysis indicated that there was no significant association between ALDH2 polymorphism, ADH2 polymorphism, or ALDH2 polymorphism–alcohol intake interaction and HCC risk in the East Asians.     

Genetic variation in DNA repair pathway genes and premenopausal breast cancer risk

Purpose We comprehensively evaluated genetic variants in DNA repair genes with premenopausal breast cancer risk. Methods In this nested case–control study of 239 prospectively ascertained premenopausal breast cancer cases and 477 matched controls within the Nurses’ Health Study II, we evaluated 1,463 genetic variants in 60 candidate genes across five DNA repair pathways, along with DNA polymerases, Fanconi Anemia complementation groups, and other related genes. Results Four variants were associated with breast cancer risk with a significance level of <0.01; two in the XPF gene and two in the XRCC3 gene. An increased risk was found in those harboring a greater number of missense putative risk alleles (a priori defined in an independent study) in the non-homologous end-joining (NHEJ) repair pathway of double-strand breaks (odds ratio (OR) per risk allele, 1.37 (95% confidence interval (CI), 1.03–1.82), P trend, 0.03). Conclusions This study implicates variants of genes in the double-strand break repair pathway in the etiology of premenopausal breast cancer. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Predicted cardiovascular mortality and reported cardiovascular morbidity in testicular cancer survivors

Introduction  We examined if testicular cancer (TC) treatment is associated with any risk for cardiovascular morbidity or predicted mortality according to the SCORE model, in which a 10-year future risk of ≥5% for developing a fatal cardiovascular event qualify for high-risk status. Methods  One thousand one hundred thirty-four TC survivors treated 1980–1994 participated in this study (1998–2002). Patients were categorised in four treatment groups: surgery (n = 225), radiotherapy (n = 445), and two chemotherapy groups: cumulative cisplatin dose ≤850 mg (n = 375) and >850 mg (cis>850, n = 89). Patients with cardiovascular disease, diabetes or SCORE ≥5% constituted a high-risk group, and those with SCORE >1% an intermediate/high risk group. Results  Age-adjusted mean SCORE was 0.93% for the surgery group. In comparison, chemotherapy treated patients had significantly higher SCORE (1.07%, p = 0.01). Only 15% of patients were scored to be at high-risk, while 53% qualified for the intermediate/high risk group. Patients in the cis>850 group had increased odds for having intermediate/high risk, compared with the surgery group (OR 3.4, 95% CI 1.3–8.7). Only 23 cardiovascular events had occurred since the testicular cancer diagnosis. Conclusion  The SCORE model indicates that patients treated with cisplatin-based chemotherapy have a significantly increased future risk of a fatal cardiovascular event. Implications for cancer survivors  TC survivors should be followed regularly with respect to cardiovascular risk profile beyond the routine 10-year clinical follow-up.     

The influence of relative body weight on toxicity of combination chemotherapy with cisplatin and etoposide

Purpose: This study was conducted to determine whether there was any relationship between the adverse toxicity of combination chemotherapy and clinical values including age, sex, creatinine clearance (Ccr), body surface area and relative body weight. Methods: Cisplatin at a dose of 80 mg/m² on day 1 and etoposide at a dose of 100 mg/m² on days 1, 2 and 3 were given to 42 consecutive patients with solid tumors. All patients had normal major organ function and received uniform hydration therapy. Results: Body Mass Index as a measure of relative body weight was inversely correlated with the percentage decrease in white blood cells (P = 0.0681) and platelet count (P = 0.0115). Body surface area was also inversely correlated with leukopenia (P = 0.0171) and thrombocytopenia (P = 0.0058). In contrast, age, sex and Ccr had no significant relationship with adverse toxicity. Conclusions: It is concluded that dose adjustment of combination chemotherapy with cisplatin and etoposide according to age or ideal body weight is not appropriate and that a conventional dose modification method based solely on body surface area is probably not sufficient to reduce interpatient variability of cancer chemotherapy. A pharmacokinetic and pharmacodynamic study of combination chemotherapy is warranted to establish the ideal dose modification method. Received: 11 September 1997 / Accepted: 9 March 1998     

Contribution of XPD (Lys751Gln) and XRCC1 (Arg399Gln) Polymorphisms in Familial and Sporadic Breast Cancer Predisposition and Survival: An Indian Report

The etiology of a significant proportion of familial breast cancers is still poorly understood, with known high penetrance gene mutations accounting for only a small proportion of the cases. The increased risk of breast cancer for the majority of women with a family history likely reflects shared minor low penetrant genetic factors. In the present case-control study undertaken to examine the influence of DNA damage repair gene polymorphisms in familial and sporadic breast cancer susceptibility, 219 Sporadic and 140 familial breast cancer patients and 367 controls were genotyped using PCRRFLP. Odds Ratios (ORs) and 95% Confidence Intervals (95%CIs) were calculated by unconditional logistic regression adjusted to age. Variant genotypes XRCC1 Arg/Gln or Gln/Gln and XPD Lys/Gln or Gln/Gln increased both familial and sporadic breast cancer susceptibility. However, when the intra group risk was compared, the risk due to the XPD polymorphic genotypes Lys/Gln or Gln/Gln was significantly lower among familial breast cancer patients compared to sporadic breast cancer patients [OR = 0.61; 95%CI = 0.39–0.94; p value = 0.024) whereas the risk implied by XRCC1 variant genotype was not significantly different between the familial and nonfamilial groups of breast cancer patients [OR = 0.97; 95%CI = 0.63–1.49; p value = 0.882]. Both these variant genotypes were not associated with the disease characteristics or survival of either familial or sporadic breast cancer patients. This study represents an addition to previous published work on GSTs from the same study population and substantiates the hypothesis that the impact of the low penetrance gene polymorphisms differ by family history of the disease.     

Four-hourly scheduling of temozolomide improves tumour growth delay but not therapeutic index in A375M melanoma xenografts

Purpose: To establish whether temozolomide is more effective against A375M human melanoma xenografts if given every 4 h rather than every 24 h, in order to exploit depletion of the DNA repair protein O ⁶-alkylguanine-DNA alkyltransferase (ATase) by prior doses of the drug. Methods: ATase depletion in A375M human melanoma xenografts was determined over 24 h after a single dose of temozolomide. The effect of different drug schedules (all of total dose 500 mg/kg) in delaying the growth of the xenografts was tested, and ATase depletion and DNA methylation damage assessed in tumour and normal tissue. Results: Maximal depletion of ATase in tumour, to 2.52 ± 0.23% of pretreatment levels, occurred 4–8 h after a single 100 mg/kg i.p. dose of temozolomide, with 23.0% recovery of protein levels at 24 h. Scheduling of temozolomide every 4 h increased tumour growth delay (33.6 ± 1.39 days with temozolomide 100 mg/kg 4-hourly ×5 versus 23.2 ± 1.43 days with temozolomide 100 mg/kg once daily ×5; P < 0.0001) at the expense of increased toxicity (17.4 ± 1.55% animal weight loss versus 10.6 ± 1.27%, respectively). Temozolomide every 4 h did not increase ATase depletion compared with the 5-day schedule, but resulted in greater DNA O ⁶-guanine methylation (29.0% more in tumour, 20.8% in liver and 56.0% in brain, comparing areas under the methylation-time curve). Conclusions: The 4-hourly schedule of temozolomide delayed tumour growth significantly more than the once-daily and 12-hourly schedules, probably as a result of greater DNA damage inflicted, but also increased toxicity. It remains to be seen if this regimen confers a net benefit over the standard schedule. Received: 12 March 1999 / Accepted: 9 August 1999     

Metabolism of N -[2-(dimethylamino)ethyl]acridine-4-carboxamide in cancer patients undergoing a phase I clinical trial

N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (DACA) is an experimental antitumour agent that has just completed phase I clinical trials in New Zealand and the United Kingdom. Urine (0–72 h) was analysed from 20 patients receiving DACA infused over 3 h (dose range 60–1000 mg/m², the latter being the highest dose achieved in the trial). Aliquots were analysed for DACA and its metabolites by high-performance liquid chromatography (HPLC). Over 72 h, 44 ± 5% (range 20–60%) of the dose was recovered in the urine, with 0.8 ± 0.3% (range 0–3.1%) occurring as DACA. The major urinary metabolite was DACA-N-oxide-9(10H)acridone, accounting for 34 ± 3% of the dose. Minor metabolites were identified as N-mono- methyl-DACA-9(10H)acridone (2.0 ± 0.5%), DACA-9(10H)acridone (3.3 ± 0.5%), N-monomethyl-DACA (0.2 ± 0.1%) and DACA-N-oxide (0.5 ± 0.1%). No ring-hydroxylated metabolite was detected. The urinary excretion of metabolites was greatest over 0–6 h in most patients. The composition of urinary metabolites was also independent of the delivered dose. Plasma was sampled at intervals throughout the infusion and at time points up to 48 h post-administration. The major plasma metabolites observed were DACA-9(10H)acridone and DACA-N-oxide-9(10H)acridone. These results indicate that, based on urinary excreted metabolites, the major biotransformation reactions for DACA in humans involve N-oxidation of the tertiary amine side chain and acridone formation, both of which appear to be detoxication reactions. Received: 21 August 1998 / Accepted: 10 December 1998     

Toenail selenium status and DNA repair capacity among female <Emphasis Type="Italic">BRCA1</Emphasis> mutation carriers

Selenium is an important cofactor of various antioxidant enzymes and has been shown to enhance DNA repair in normal human fibroblasts. Oral selenium supplementation has also been shown to decrease the number of chromosome breaks in BRCA1 mutation carriers. Because the predisposition to cancer among BRCA1 mutation carriers may be linked to high rates of DNA damage and chromosome breakage, we evaluated the association between toenail selenium concentrations and three measures of DNA repair capacity (the single-cell alkaline gel electrophoresis (comet) assay, the micronucleus test, and the enumeration of γ-H2AX nuclear foci) in female BRCA1 mutation carriers and in non-carriers. Toenail selenium levels were inversely associated with levels of chromosomal damage following exposure to gamma-irradiation, as assessed by the micronucleus test. This association was limited to women with a BRCA1 mutation (p = 0.03). Toenail selenium was not a significant predictor of DNA repair capacity, as quantified by either the comet assay or the number of γ-H2AX foci, in carriers or in non-carriers. These results provide evidence for a possible protective effect of selenium against BRCA1-associated breast cancers.     

Investigation of bioavailability and pharmacokinetics of treosulfan capsules in patients with relapsed ovarian cancer

Purpose: Treosulfan (l-threitol-1,4-bis-methanesulfonate, Ovastat) is a prodrug of a bifunctional alkylating agent with activity in ovarian carcinoma and other solid tumors. In a pharmacologic study of the bioavailability of treosulfan in a capsule formulation, patients with relapsed ovarian carcinoma were treated with alternating doses of oral and intravenous (i.v.) treosulfan of 1.5 or 2.0 g daily for 5 to 8 days. Methods: A sensitive method for the determination of treosulfan in plasma and urine by reversed-phase high-performance liquid chromatography had previously been developed. Pharmacokinetic analyses of treosulfan were carried on plasma and urine samples from 20 i.v. courses and 20 courses of oral administration. Results: The bioavailability ratio (f) of oral to i.v. administration was calculated as 0.97 ± 0.18 (mean ± SD) using the values AUC_oral=82.1 ± 39.4 μg/ml h and AUC_i.v.=85.4 ± 30.3 μg/ml h. The peak plasma concentration c_max (29 ± 14 μg/ml vs 65 ± 23 μg/ml) was significantly (P < 0.01) higher after i.v. administration and the t_max after oral administration was 1.5 ± 0.34 h. The terminal half-life of treosulfan was about 1.8 h. The mean urinary excretion of the parent compound was about 15% of the administered total dose over 24 h (range 6–26%). Conclusions: The high and relatively constant bioavailability of treosulfan indicates that capsules provide a satisfactory noninvasive treatment alternative. A feasible and reliable oral treosulfan formulation could provide the basis for the development of long-term low-dose outpatient treatment of patients with malignant diseases. Received: 28 July 1999 / Accepted: 16 December 1999     

N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine (DPPE), a chemopotentiating and cytoprotective agent in clinical trials: interaction with histamine at cytochrome P450 3A4 and other isozymes that metabolize antineoplastic drugs

Purpose: N,N-diethyl-2-[4-(phenylmethyl)phe- noxy]ethanamine · HCl (DPPE), an intracellular histamine (HA) antagonist with chemopotentiating and cytoprotective properties, is currently in phase 2 and 3 clinical trials in breast and prostate cancer. DPPE modulates growth at in vitro concentrations that antagonize HA binding to cytochromes P450 in rat liver microsomes. HA inhibits P450 metabolism of some drugs. Recent in vitro studies in human colon cancer cells have linked DPPE enhancement of paclitaxel, doxorubicin and vinblastine cytotoxicity to inhibition of the P-glycoprotein (P-gp) pump. Many substrates of P-gp are also substrates of CYP3A4, a P450 isozyme that metabolizes a variety of antineoplastic agents and is highly expressed in some malignant tissues. Therefore, we assessed whether (a) DPPE and HA interact at CYP3A4 and other P450 human isozymes, and (b) DPPE inhibits the catalytic activity of CYP3A4. Methods: Using spectral analysis, we measured DPPE and HA binding to insect microsomes that express human P450 isozymes 1A1, 2B6, 2D6 or 3A4. Employing thin-layer chromatography, we assessed the metabolism of DPPE by each isozyme and DPPE inhibition of testosterone metabolism by CYP3A4 and by rat liver microsomes. Results: (1) DPPE evoked “type I” (substrate site binding) absorbance-difference spectra with CYP2D6 (K_s=4.1 ± 0.4 μM), CYP3A4 (K_s= 31 ±15 μM) and CYP1A1 (K_s=40 ± 9 μM), but not with CYP2B6. (2) In correspondence with the binding studies, DPPE was metabolized by CYP2D6, CYP3A4 and CYP1A1; no metabolism occurred with CYP2B6. (3) HA evoked “type II” (heme iron binding) absorbance-difference spectra with all four isozymes, with K_s values in the range 80–600 μM. DPPE inhibited HA (600 μM) binding to CYP2D6 (IC₅₀=4 μM, 95% CI=1.8–8.9 μM) and CYP1A1 (IC₅₀=135 μM: 95% CI=100–177 μM), but stimulated HA (500 and 1000 μM) binding to CYP3A4 (EC₅₀=155 μM, 95% CI=104–231 μM). DPPE did not affect HA binding to CYP2B6. (4) DPPE inhibited the metabolism of testosterone by CYP3A4. The concentration/effect curve was biphasic: DPPE inhibited metabolism by 30% at the first site (IC₅₀=3 μM, 95% CI=0.5–25.5 μM), and an additional 70% inhibition occurred at the second site (IC₅₀=350 μM, 95% CI=215–570 μM). A similar result was observed with rat liver microsomes. Conclusion: DPPE is a substrate for CYP3A4, CYP2D6 and CYP1A1, but not CYP2B6. DPPE inhibits testosterone metabolism by interacting at two sites on CYP3A4, the first correlating with its K_s value to bind the substrate site and the second, with its EC₅₀ value to enhance HA binding to the heme iron. We postulate that (1) the inhibitory effect of DPPE on CYP3A4 activity is mediated directly at the substrate site and indirectly by its enhancement of the binding of HA to the heme moiety; (2) in tumor cells that express high constitutive levels of CYP3A4, potentiation of chemotherapy cytotoxicity by DPPE results, in part, from inhibition of CYP3A4-mediated metabolism and P-gp-mediated efflux of antineoplastic drugs; (3) in normal cells that express low constitutive levels of the isozyme, cytoprotection by DPPE results, in part, from induction of CYP3A4 and P-gp, resulting in an increase both in metabolism and efflux of antineoplastic drugs. Received: 26 April 1999 / Accepted: 3 September 1999     

Comorbidities, therapy, and newly diagnosed conditions for women with early stage breast cancer

Purpose  To describe comorbidities in breast cancer patients at diagnosis and examine factors associated with self-reported comorbidities 30 months post-diagnosis. Methods  Nine hundred forty one of 1,171 women had a medical record abstract and a follow-up survey in the Health, Eating, Activity and Lifestyle Study. Results  We compared our breast cancer cohort to a contemporaneous nationally-representative sample of age, race/ethnicity and education matched women without cancer (n = 865). Breast cancer patients did not have substantially more comorbidities than women without breast cancer. Women with a hospital record of congestive heart failure significantly less often received chemotherapy or radiation following breast conserving surgery. In multivariate analysis, women who received chemotherapy alone (OR = 3.2; 95% CI: 1.5–6.8), chemotherapy plus radiation (OR = 1.9; 95% CI: 1.02–3.7) or radiation plus tamoxifen (OR = 1.9; 95% CI: 1.1–3.2) were significantly more likely to report at least one new comorbid condition following breast cancer diagnosis than women who received no chemotherapy, tamoxifen or radiation. Overall, women who received adjuvant therapy were more likely to have new comorbidities. Conclusions  Comorbidities were not substantially different in breast cancer patients than the non-cancer matched controls. Future research should focus on efforts to minimize comorbidities related to chemotherapy and other combination therapy. Funding source  N01-PC-35139, N01-PC-35142, N01-PC-35138     

Chemotherapy response analysis for osteosarcom with intra-arterial chemotherapy by subcutaneous implantable delivery system

To summarize the experience in intraarterial neoadjuvant chemotherapy for extremity osteosarcoma. Between January 2002 and December 2007,111 patients with stage IIB extremity osteosarcoma received preoperative intraarterial therapy with subcutaneous implantation of chemotherapy pump as well as en bloc resection, and postoperative adjuvant chemotherapy. There were 63 males and 48 females with an average age of 18 (range, 14 ~ 39 years). The time from symptom onset to hospitalization varied from several days to 6 months. The induction chemotherapy regimen includes: epirubicin [50 ~ 70 mg/m² by 4-hour intraarterial infusion/day for 3 day] and cisplatin [100 ~ 120 mg/m² by 2-hour intraarterial infusion/day for 3 days] repetitively every 2 ~ 3 weeks. Among which 24 cases only received two cycles induction chemotherapy was set to nonstandard chemotherapy group and 87 cases received three to six cycles induction chemotherapy set to standard chemotherapy group. The number of preoperative chemotherapy-cycles of standard chemotherapy group depends on the clinical and radiographic evaluation of chemotherapy efficacy. Median follow-up time was 28(8 ~ 48) months. The rate of limb preservation surgery was 89.53% (77/86) in standard chemotherapy group,and was 37.5% (9/24) in nonstandard chemotherapy group. Kaplan-Meier survival analysis showed that the 3-year overall survival rate and disease free survival rate of all the 111 cases were 68.3% and 65.9% respectively. There were significant differences in overall survival rate (38.9%, 80.0%, P = 0.000), disease free survival rate (30.1%, 79.5%, P = 0.000), distant metastasis rate (66.67%, 16.09%, P = 0.0000) and local recurrence rate (58.33%, 13.79%, P = 0.0000) between nonstandard chemotherapy group and standard chemotherapy group. Standard intraarterial neo-adjuvant chemotherapy was more effective than nonstandard intraarterial induction chemotherapy to stage IIB extremity osteosarcoma.     

Pharmacologic effects of paclitaxel in human bladder tumors

Purpose: The goal of this study was to determine whether paclitaxel, when given by a 2-h treatment, produces significant cytotoxic effects in human bladder transitional cell carcinoma and hence qualifies as a candidate drug for intravesical treatment. Methods: Histocultures of surgical specimens from patients (n = 16) were used. Results: Paclitaxel produced partial inhibition of DNA precursor incorporation in about 70% of tumors and induced apoptosis in about 90% of tumors, while these effects were minimal or not detectable in the remaining tumors. In the responsive tumors, the average maximal inhibition of DNA synthesis was 60% and the average maximal apoptotic index was 15%. Resistance to antiproliferative and apoptotic effects was not always found in the same individual tumors, and no relationship was found between the magnitude of antiproliferative and apoptotic effects in individual tumors. The maximal apoptotic index correlated with the LI for the untreated control (r ² = 0.42, P < 0.01). More than 95% of apoptotic cells were labeled by DNA precursor, whereas not all labeled cells were apoptotic. The pharmacologic effects of paclitaxel in bladder tumors were qualitatively equivalent to those previously found in human head and neck tumors and in human prostate tumors after treatment for longer periods of 24 to 96 h. Conclusions: These results indicate that a 2-h paclitaxel treatment was sufficient to produce antiproliferation and apoptosis in 70–90% of human bladder tumors, and the apoptotic effect appeared to be linked to proliferation and occurred after DNA synthesis. Received: 19 December 1996 / Accepted: 20 March 1997     

Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells

Purpose: We have reported previously that the expression of E. coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity. In the study reported here, we further characterized the ability of dutE expression in one HT29 clone, dutE7, to alter the effects of treatment with FdUrd and other thymidylate synthase (TS) inhibitors. In addition, we developed two HuTu80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytotoxicity. Methods: Both a dutE retroviral expression system and a dutE antibody were developed to facilitate the generation and screening of dutE-expressing clones. HT29 and HuTu80 clones expressing dutE were tested for drug-induced DNA damage with either alkaline elution or pulsed field gel electrophoresis and drug-induced loss of clonogenicity. Results: Following a 24-h treatment with 100 μM CB3717 or 500 nM methotrexate (MTX), dutE7 cells were significantly less sensitive to drug-induced loss of clonogenicity than con3 cells. DutE7 cells were also resistant to CB3717-induced DNA fragmentation at 24 h. However, following a 48-h treatment with CB3717 or MTX there was no difference in survival between con3 and dutE7 cells, even though DNA damage was still greatly attenuated in the dutE7 cell line. In addition, expression of dutE in two HuTu80 clones, 80  C and 80  K, did not protect these cells from FdUrd-induced DNA damage or cytotoxicity. Conclusions: We conclude that the role of uracil misincorporation and subsequent DNA damage in cytotoxicity induced by TS inhibitors, in HT29 cells, is time dependent, and that cytotoxicity caused by long-term exposure to these drugs is largely independent of resultant DNA damage, in this cell line. The inability of dutE to protect HuTu80 cells from FdUrd further suggests that the significance of uracil misincorporation resulting from TS inhibition varies among cell lines. Received: 19 August / Accepted: 16 December 1997     

A phase II pharmacodynamic study of pyrazoloacridine in patients with metastatic colorectal cancer

Purpose: To perform a phase II trial of pyrazoloacridine (PZA), a novel DNA intercalator, in patients with metastatic colorectal carcinoma and no previous therapy. Methods: PZA was administered at a dose of 750 mg/m² intravenously over 3 h every 21 days. Pharmacokinetic studies to determine PZA plasma concentrations were performed. Results: No responses were seen in 14 response-evaluable patients. Patients received a median of two cycles of PZA (range 1–6). Toxicity included neutropenia and neurologic side-effects, which were ≥ grade III in 73% and 14%, respectively. High plasma concentrations of PZA (C_max) correlated with low neutrophil counts (P=0.04). Conclusions: PZA is inactive at this dose and schedule in colorectal cancer, and produces moderately severe toxicity. Received: 18 October 1999 / Accepted: 16 March 2000