Chapter 27
Detection of Cisplatin—DNA Adducts in Humans 1
1
2
Miriam C. Poirier , Shalina Gupta-Burt , Charles L. Litterst , and Eddie Reed Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 11, 2018 at 03:15:44 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
1
1
2
National Cancer Institute and National Institute of Arthritis and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
A n E L I S A , which measures cisplatin-DNA intrastrand adducts, and atomic absorbance spectrometry, which measures total platinum bound to D N A , have been used to quantify DNA modification in samples from patients receiving platinum drug-based therapy and rats in which the treatment of human cancer patients has been modeled. Adducts measured in blood cell DNA samples from cancer patients have correlated with dose and chemotherapeutic efficacy. Human tissue D N A adducts have a widespread distribution, and long-term adduct persistence (> 1 year) has been observed in many organs including tumor and target sites for drug toxicity. In recent years a large number of polyclonal and monoclonal antisera have been produced against adducts or modified D N A samples of a variety of chemical classes, including methylating and ethylating agents, aromatic amines, polycyclic aromatic hydrocarbons (PAH), aflatoxins, psoralens and platinum-ammine complexes (1.2). These antisera have been used to establish highly-sensitive quantitative immunoassays and have been adapted for immunohistochemistry, and electron microscopy. The determination of carcinogen-DNA adduct levels by quantitative immunologic procedures has certain advantages over other techniques (1). The sensitivity is frequently better than that obtained with radiolabeled carcinogens. Carcinogen-DNA adduct antisera generally do not react with structurally-dissimilar adducts of the same carcinogen, the carcinogen alone, unmodified nucleosides or unmodified D N A . They are therefore highly-specific probes for chemical bound to D N A . Immunologic assays are rapid, highly reproducible, and can be used in many situations where the cost of a radiolabeled compound would be prohibitive; for example, long-term chronic administration of a carcinogen. In addition, because these techniques are highly sensitive they have been successfully applied to the determination of D N A adducts in tissues of humans exposed to chemicals (2). Cisplatin is a chemotherapeutic agent responsible for the cure of testicular cancer, and in widespread use for ovarian, head and neck and lung
This chapter not subject to U.S. copyright Published 1991 American Chemical Society
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
27.
POIRIER ET AL.
Detection of Cisplatin-DNA
Adducts in Humans
301
cancers. Because it is a rodent carcinogen (2) and causes the f o r m a t i o n of D N A adducts it was o f interest for us to investigate D N A adducts formed by this drug i n tissues o f h u m a n cancer patients receiving p l a t i n u m drug chemotherapy. Efforts have been made to correlate the extent o f cisplatinD N A adduct formation with dose, toxicity and response of the disease to chemotherapy. T h e U s e o f E n z y m e - L i n k e d Immunosorbent Assay ( E L I S A ) and A t o m i c A b s o r b a n c e Spectrometry ( A A S ^ for D e t e r m i n a t i o n of C i s p l a t i n - D N A Adducts. D N A modified with the chemotherapeutic agent cisplatin (tis-diammined i c h l o r o p l a t i n u m [II]) has b e e n used to elicit antisera specific for the intrastrand bidentate N 7 - d ( G p G ) - and N 7 - d ( A p G ) - d i a m m i n e p l a t i n u m adducts (4). These adducts comprise a major p o r t i o n (80%) of p l a t i n u m b o u n d to D N A i n biological samples from cells, animals or humans receiving p l a t i n u m drug exposure (5.6). T h e antiserum recognizes modified D N A samples and is not specific for the individual adducts alone, u n m o d i f i e d D N A or u n m o d i f i e d nucleotides (4). It has been demonstrated (7) that the E L I S A , as performed w i t h a calf thymus D N A standard modified 1-4%, similar to the i m m u n o g e n D N A , substantially underestimates the total intrastrand adducts i n a biological D N A sample. This is because the three-dimensional conformation i n a D N A modified i n the range of several adducts per 100 nucleotides is significantly different from a biological sample D N A modified i n the range o f one adduct i n a m i l l i o n nucleotides. T h e r e is, however, good correlation between biologically-relevant events such, as dose response and disease response, and D N A adducts determined by the E L I S A . Thus the assay measures D N A damage i n an internally-consistent fashion, even though the values do not reflect the total number of adducts. In order to obtain quantitative evaluation of cisplatin b o u n d to D N A , we now assay a l l h u m a n samples by both E L I S A and A A S . Because at least 8 0 % o f the total p l a t i n u m b o u n d to D N A i n a biological sample is i n the form of intrastrand adducts, A A S gives values which are close to the desired quantitation. A A S with Z e e m a n background correction has sufficient sensitivity to measure cisplatin i n biological samples (8). I n order to compare results w i t h the two methods, 44 samples of rat tissue were assayed by b o t h E L I S A and A A S , and w h e n data from the two methods were plotted simultaneously (Figure 1), the correlation observed was essentially linear. T h e actual numbers generated by the A A S were approximately 500 times higher than those obtained by E L I S A , as shown i n the Figure. M o n i t o r i n g of C i s p l a t i n - D N A A d d u c t s i n B l o o d C e l l D N A of C a n c e r Patients T h e E L I S A , described above, has been utilized to measure D N A adducts i n nucleated b l o o d cell D N A and tissue D N A of cancer patients receiving p l a t i n u m drug-based chemotherapy. Advantages o f this approach include the opportunity to obtain h u m a n samples from unexposed controls, and the potential to observe a dose-response for adduct formation since precise dose information is available. I n addition, it was anticipated that i f adducts could
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
302
IMMUNOASSAYS FOR TRACE CHEMICAL ANALYSIS
be correlated w i t h biological effects such as disease response or toxicity, any manipulations w h i c h w o u l d enhance adduct formation might actually improve therapy. B l o o d cell D N A samples were obtained from testicular and ovarian cancer patients receiving p l a t i n u m drug-based therapy during 5 day drug infusions at the N I H C l i n i c a l C e n t e r (9), and assayed for adducts by cisplatinD N A E L I S A . These studies demonstrated the first dose-response for D N A adduct formation i n humans (Figure 2), and samples from unexposed controls were consistently negative. A b o u t half of the treated patients experienced a dose-related increase i n adducts w i t h subsequent monthly cycles of chemotherapy, and the other half d i d not have measurable adducts even after very high total cumulative doses (9). T h i s suggests that there may be b i o c h e m i c a l or metabolic factors which govern interindividual variability i n D N A adduct processing. W h e n adducts were correlated with disease response i n 72 ovarian and poor-prognosis testicular cancer patients (Figure 3), a trend was observed i n w h i c h the complete responders had the highest m e a n adduct levels and D N A from many of the non-responders d i d not contain measurable adducts (10.11). T h e trend analysis, which compared m e a n adduct levels for complete responders, partial responders and non-responders was significant with a Ρ value of 0.03 (10). These data support the contention that the formation of high levels of the c i s p l a t i n - D N A intrastrand adducts, as measured by the E L I S A , is associated w i t h successful chemotherapy, and that those individuals who fail to form measurable adducts have a high rate of therapy failure. In an extension of these studies (12) a series of prognostic variables, including D N A adduct formation, Karnofsky status, total cumulative p l a t i n u m dose, stage of disease, b u l k of disease at initiation of therapy and histologic type and grade, was correlated with disease response by univariate and multivariate analysis. In an analysis of data from 24 ovarian cancer patients the only variable to be significantly associated with disease response was c i s p l a t i n - D N A adduct formation measured by the E L I S A . M o n i t o r i n g o f C i s p l a t i n - D N A A d d u c t s i n Tissue D N A o f C a n c e r Patients E v e n though b l o o d cell D N A adducts correlate with platinum-drug dose i n some individuals, and are associated with a positive response to therapy, experiments with b l o o d cell D N A do not indicate the extent of adduct formation i n the target tumor tissue or i n organs w h i c h undergo toxicity. I n order to investigate D N A adduct formation i n tumor and other h u m a n tissues, adducts were measured i n tumor biopsies and i n tumor tissues obtained at autopsy. I n one study, biopsies of cervical tumor were taken before and 24 hr after treatment with carboplatin (7). In addition, b l o o d was drawn from the same individuals before, 24 hr after and 8 days after chemotherapy. D N A adducts, measured by E L I S A (Table I), were similar i n tumor and i n b l o o d 24 hr after the drug infusion, and continued to be elevated i n b l o o d 8 days later. T h e results demonstrated that adduct levels i n b l o o d cell D N A were reflective of those i n cervical tumor D N A after a single carboplatin dose.
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
27.
POIRIER ET A L
Detection of Cisplatin-DNA
Adducts in Humans
303
800
600 h D
TJ Ό
