carboplatin-DNA Adducts Research Articles

A diagnostic microdosing approach to investigate platinum sensitivity in non-small cell lung cancer.

The platinum-based drugs cisplatin, carboplatin and oxaliplatin are often used for chemotherapy, but drug resistance is common. The prediction of resistance to these drugs via genomics is a challenging problem since hundreds of genes are involved. A possible alternative is to use mass spectrometry to determine the propensity for cells to form drug-DNA adducts-the pharmacodynamic drug-target complex for this class of drugs. The feasibility of predictive diagnostic microdosing was assessed in non-small cell lung cancer (NSCLC) cell culture and a pilot clinical trial. Accelerator mass spectrometry (AMS) was used to quantify [14 C]carboplatin-DNA monoadduct levels in the cell lines induced by microdoses and therapeutic doses of carboplatin, followed by correlation with carboplatin IC50 values for each cell line. The adduct levels in cell culture experiments were linearly proportional to dose (R2 = 0.95, p < 0.0001) and correlated with IC50 across all cell lines for microdose and therapeutically relevant carboplatin concentrations (p = 0.02 and p = 0.01, respectively). A pilot microdosing clinical trial was conducted to define protocols and gather preliminary data. Plasma pharmacokinetics (PK) and [14 C]carboplatin-DNA adducts in white blood cells and tumor tissues from six NSCLC patients were quantified via AMS. The blood plasma half-life of [14 C]carboplatin administered as a microdose was consistent with the known PK of therapeutic dosing. The optimal [14 C]carboplatin formulation for the microdose was 107 dpm/kg of body weight and 1% of the therapeutic dose for the total mass of carboplatin. No microdose-associated toxicity was observed in the patients. Additional accruals are required to significantly correlate adduct levels with response.

Microdose-Induced Drug-DNA Adducts as Biomarkers of Chemotherapy Resistance in Humans and Mice.

We report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [14C]carboplatin (1% of the therapeutic dose). Carboplatin-DNA adducts were quantified by accelerator mass spectrometry in blood and tumor samples collected within 24 hours, and compared with subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice were dosed with [14C]carboplatin or [14C]gemcitabine and the resulting drug-DNA adduct levels were compared with tumor response to chemotherapy. At least one of the drugs had to induce high drug-DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug-DNA adducts as predictive biomarkers. Mol Cancer Ther; 16(2); 376-87. ©2016 AACR.

Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity.

This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 10(8) nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 10(8) nucleotides per hour in carboplatin alone (p = 0.021). This rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.

A microdosing study to identify chemoresistance in bladder cancer.

356 Background: DNA adduct formation and incorporation of gemcitabine into genomic DNA are critical steps in cancer cell response to platinum (Pt) and gemcitabine chemotherapy, respectively. We hypothesize that levels of Pt-DNA adducts and gemcitabine in genomic DNA below a threshold are predictive of chemoresistance. Accelerator mass spectrometry (AMS) is an ultrasensitive method for measuring radiocarbon. By measuring 14C bound to DNA, AMS was used to quantify carboplatin-DNA damage and gemcitabine incorporation into DNA after mice or patients received nontoxic “microdoses” of 14C-labeled carboplatin or gemcitabine. Methods: Cancer cells and mice bearing tumor xenografts were treated with one microdose (1% of the therapeutic dose) or therapeutic dose of [14C]carboplatin or [14C]gemcitabine. Carboplatin-DNA adducts and gemcitabine incorporation in DNA were correlated with cell/tumor response to chemotherapy. In the Phase 0 trial, patients with advanced bladder or non-small cell lung cancer were treated with one microdose of [14C]carboplatin followed by tumor sampling 24 hours later. Carboplatin-DNA adducts and other relevant parameters, such as pharmacokinetics and repair of DNA damage, were measured and correlated with cancer response to chemotherapy. Results: The levels of microdose-induced carboplatin-DNA damage were linearly proportional to that caused by the therapeutic dose (R2=0.92, p&lt;0.001); and correlated with chemoresistance to carboplatin. Low gemcitabine incorporation into DNA correlated to gemcitabine resistance in patient-derived bladder cancer xenografts (p&lt;0.001). In the Phase 0 trial, 18 patients have been enrolled. DNA damage induced by carboplatin was measured by AMS in peripheral blood mononuclear cells and, in some patients, in tumor tissues; and will be correlated with cancer response to chemotherapy. The pharmacokinetics and DNA adduct levels were linear between microdose and therapeutic doses. No microdose-related toxicity was observed. The radiation exposure was less than that of one chest X-ray per microdose. Conclusions: The levels of DNA damage induced by nontoxic microdosing carboplatin can potentially predict chemoresistance. The current status of the Phase 0 trial will be presented. Clinical trial information: NCT01261299.

Abstract 1183: Phase 0 microdosing trial quantitating DNA adduct formation to identify platinum chemosensitivity: from bench to bedside.

Abstract Purpose: DNA adduct formation is a critical step in platinum(Pt)–induced cell death. The goal of this project is to measure carboplatin-DNA adducts after a single non-toxic microdose of [14C]carboplatin, use this information to predict efficacy before cancer patients receive toxic chemotherapy and assess the underlying chemoresistance mechanisms, thus enhancing personalized therapy. Methods: Six non-small cell lung cancer (NSCLC) and six bladder cancer cell lines were treated with 1 μM (microdose) or 100 μM (therapeutic dose) carboplatin, but with equal amounts of [14C]carboplatin. Carboplatin-DNA monoadduct levels were determined by measuring 14C content in genomic DNA with accelerator mass spectrometry (AMS). Cellular sensitivity to carboplatin (IC50 values) was correlated with drug-DNA adduct levels. To validate the in vitro findings, a pilot Phase 0 trial with microdosing [14C]carboplatin was initiated to determine the recommended Phase II dose (RP2D) of [14C]carboplatin for optimal measurement of DNA monoadducts and pharmacokinetics (PK) in lung and bladder cancer. PK and DNA monoadduct levels in peripheral blood mononuclear cells and tumor tissues were determined by liquid scintillation counting and AMS, respectively, validated with inductively coupled plasma mass spectrometry (ICP-MS), and correlated with tumor response to chemotherapy. Results: DNA monoadduct levels in cell lines induced by microdose and therapeutic concentrations of carboplatin were highly linearly proportional (r2 = 0.95, p &amp;lt; 0.001) as measured by AMS, and correlated to the total Pt-DNA adduct levels induced by therapeutic carboplatin (r2 = 0.80, p &amp;lt; 0.001) as measured by ICP-MS. DNA adduct levels were superior in predicting resistance to carboplatin when compared to the mRNA expression of ERCC1, a DNA repair gene commonly used as a resistant biomarker of Pt-based chemotherapy. Nine patients were recruited at the pilot Phase 0 trial. The Level I dose of [14C]carboplatin at 107 dpm/Kg with total carboplatin at 1% of therapeutic dose (area under curve of 6) was determined as the RP2D. No clinical toxicity was observed as a result of microdosing. The radiation exposure from 14C was less than a chest X-ray per patient. DNA monoadduct levels and PK parameters of carboplatin at the microdose and therapeutic doses were highly correlated (p&amp;lt;0.001), suggesting that this non-toxic microdosing approach can be used to predict DNA adduct levels and PK of therapeutic doses. Patients with the highest DNA monoadduct levels responded best to carboplatin-based chemotherapy, although more data is needed to achieve statistical significance in the clinical data. Conclusions: This novel microdosing approach predicts DNA monoadduct levels and PK of therapeutic carboplatin dosing; preliminary data suggest the feasibility of predicting chemosensitivity and the possibility of guiding personalized dosing. Citation Format: Chong-xian Pan, Sisi Wang, Miaoling He, Tao Li, Tzu-yin Lin, Hongyong Zhang, Michael Malfatti, David Gandara, Tianhong Li, Ken Yoneda, Joyce Lee, Marc Dall'Era, George Cimino, Ralph de Vere White, Kenneth Turteltaub, Paul Henderson. Phase 0 microdosing trial quantitating DNA adduct formation to identify platinum chemosensitivity: from bench to bedside. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1183. doi:10.1158/1538-7445.AM2013-1183

Abstract LB-403: A Phase 0 microdosing clinical trial to identify chemoresistance

Abstract BACKGROUND: As alkylating agents, platinum (Pt) analogs kill cancer cells mainly through induction of DNA damage. We hypothesize that low levels of Pt-induced DNA damage are predictive of chemoresistance. Accelerator mass spectrometry (AMS) is an ultrasensitive method for measuring radiocarbon with 10−18∼21 mole sensitivity. By measuring 14C bound to DNA, AMS detects carboplatin-induced DNA damage after patients receive one subtoxic microdose of 14C-labeled carboplatin. METHODS: Cancer cells, mice bearing tumor xenografts and human cancer patients were treated with one microdose (1/100th the therapeutic dose) of [14C]carboplatin. Carboplatin-DNA adducts and other relevant parameters such as pharmacokinetics (PK), drug influx/efflux, intracellular drug inactivation, and repair of DNA damage, were measured and correlated with response to chemotherapy. RESULTS: In preclinical studies, the levels of microdose-induced DNA damage were linearly proportional to those caused by the therapeutic drug dose (R2=0.92, p&amp;lt;0.0001). When compared to the expression of ERCC1 (a gene commonly used to predict chemoresistance in non-small lung cancer in clinic), DNA damage levels were superior in predicting chemoresistance in cell lines. Measuring drug uptake/efflux, intracellular inactivation and DNA repair allowed insight into some of the resistance mechanisms. We have opened a Phase 0 clinical trial because of the microdosing approach of the study. This trial takes a two-stage design. The first stage is similar to a Phase I trial to define the recommended Phase II dose (RP2D) of 14C-carboplatin. The second stage is similar to a Phase II trial to determine if low levels of microdosing carboplatin-induced DNA damage correlate with chemoresistance and determine some of the underlying chemoresistant mechanisms. So far, we have finished the first (Phase I) stage of the Phase 0 trial. One microdose of 14C-carboplatin was administered to human cancer patients. Pt-induced DNA damage and repair, and PK were measured and correlated with the response and toxicity of chemotherapy. The RP2D of 14C-carboplatin is 107 dpm/kg of body weight, less than 1% of the radiation exposure from an abdominal CT scan. The RP2D of carboplatin is 1% of therapeutic dose. No toxicity was observed. The PK of microdosing carboplatin was the same of the therapeutic dose. Slow drug metabolism correlated with high DNA damage. Molecular analysis of genes such as ERCC1 and RRM1 is being analyzed and will be compared with the Phase 0 results. All these results will be correlated with cancer response and patients' toxicity to chemotherapy. A parallel Phase 0 clinical trial in dogs with naturally occurring cancer will also be presented. CONCLUSION: The levels of DNA damage induced by nontoxic microdosing carboplatin can potentially predict chemoresistance in cancer cell lines. The microdosing trial is nontoxic to patients. The second stage of the trial is currently going on. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-403. doi:10.1158/1538-7445.AM2011-LB-403

A phase 0 microdosing trial to identify chemoresistance in bladder cancer.

264 Background: DNA damage is the critical step in cancer cell response to platinum (Pt) chemotherapy. We hypothesize that low levels of Pt-induced DNA damage are predictive of chemoresistance. Accelerator mass spectrometry (AMS) is an ultrasensitive method for measuring radiocarbon. By measuring 14C bound to DNA, AMS can detect carboplatin-induced DNA damage after patients receive one subtoxic microdose of 14C-labeled carboplatin. Methods: Cancer cells and mice bearing tumor xenografts were treated with one microdose (1/100th of the therapeutic dose) or one therapeutic dose of [14C]carboplatin. Carboplatin-DNA adducts and other relevant parameters such as drug influx/efflux, intracellular drug inactivation, and repair of DNA damage, were measured and correlated with response to chemotherapy. Results: AMS detected Pt-DNA damage when cancer cells and mice with tumor xenografts were exposed to one microdose of [14C]carboplatin. The levels of microdose-induced DNA damage were linearly proportional to the DNA damage caused by the therapeutic drug dose (R2=0.92, p&lt;0.001); and these levels of DNA damage correlated with chemoresistance. Low DNA damage predicts chemoresistance. Measuring drug uptake/efflux, intracellular inactivation and DNA repair allowed insight into some resistance mechanisms. We have opened a phase 0 microdosing trial to study patients with bladder cancer who are scheduled to receive Pt-based chemotherapy. One subtoxic microdose of 14C-carboplatin will be administered to these patients before biopsy. Pt-induced DNA damage and repair in left-over tumor biopsy specimens and other relevant parameters will be measured and correlated with the response and toxicity of chemotherapy. Molecular analysis of genes such as ERCC1 and RRM1 will be analyzed and compared with this phase 0 results. We have opened a similar phase 0 trial in dog patients with bladder cancer. Conclusions: The levels of DNA damage induced by nontoxic microdosing carboplatin can potentially predict chemoresistance in cancer cell lines. The clinical data of the phase 0 trial will be presented. No significant financial relationships to disclose.

Gemcitabine Causes Minimal Modulation of Carboplatin-DNA Monoadduct Formation and Repair in Bladder Cancer Cells

We are developing a method to identify cellular resistance to carboplatin by using accelerator mass spectrometry to measure carboplatin-DNA adducts formed from drug microdoses (∼1/100th the therapeutic dose). Such an approach would be particularly useful if it is still valid in combination chemotherapy. We examined whether the addition of gemcitabine, another chemotherapeutic drug, could influence carboplatin-DNA adduct levels. There were no substantial differences in the levels of carboplatin-DNA adducts in cells upon exposure to the carboplatin/gemcitabine combination at various doses and schedules. These data demonstrate that microdosing is feasible for the characterization of carboplatin resistance when given in combination with gemcitabine.

Abstract 3522: A Phase 0 microdosing trial to identify chemoresistance: From bench to bedside

Abstract BACKGROUND: DNA damage is the critical step in cancer cell response to platinum (Pt) chemotherapy. We hypothesize that low levels of Pt-induced DNA damage are predictive of chemoresistance. Accelerator mass spectrometry (AMS) is an ultrasensitive method for measuring radiocarbon. By measuring 14C bound to DNA, AMS can detect carboplatin-induced DNA damage after patients receive one subtoxic microdose of 14C-labeled carboplatin. METHODS: Cancer cells and mice bearing tumor xenografts were treated with one microdose (1/100th of the therapeutic dose) or one therapeutic dose of [14C]carboplatin. Carboplatin-DNA adducts and other relevant parameters such as drug influx/efflux, intracellular drug inactivation, and repair of DNA damage, were measured and correlated with response to chemotherapy. RESULTS: AMS detected Pt-DNA damage when cancer cells and mice with tumor xenografts were exposed to one subtoxic microdose of [14C]carboplatin. The levels of microdose-induced DNA damage were linearly proportional to the DNA damage caused by the therapeutic drug dose (R2=0.92, p&amp;lt;0.001); and these levels of DNA damage correlated with chemoresistance. Low DNA damage predicts chemoresistance. Measuring drug uptake/efflux, intracellular inactivation and DNA repair allowed insight into some resistance mechanisms. A Phase 0 microdosing trial has been designed to study patients with non-small cell lung cancer or bladder cancer who are scheduled to receive Pt-based chemotherapy. One subtoxic microdose of 14C-carboplatin will be administered to these patients before biopsy. Pt-induced DNA damage and repair in left-over tumor biopsy specimens and other relevant parameters will be measured and correlated with the response and toxicity of chemotherapy. Molecular analysis of genes such as ERCC1 and RRM1 will be analyzed and compared with this Phase 0 results. CONCLUSION: The levels of DNA damage induced by nontoxic microdosing carboplatin can potentially predict chemoresistance in patients. A phase 0 microdosing trial is under the IRB review to identify chemoresistance and determine the underlying resistant mechanisms for personalized therapy before patients receive toxic Pt chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3522.

The formation and persistence of carboplatin-DNA adducts in rats.

The formation and persistence of platinum-DNA adducts were studied with immuno(cyto)chemical methods in male and female Sprague-Dawley rats treated with a single i.p. dose of carboplatin. Linear dose-effect curves were observed for kidney and liver with an immunocytochemical assay using NKI-A59 antiserum that recognizes intrastrand cross-links. With this method, no staining of the nuclei due to platinum-DNA damage could be observed in the spleen, testis, uterus, or ovary after administration of up to 80 mg/kg carboplatin. A homogeneous staining of the nuclei in the liver was observed. The nuclear staining in the kidney was somewhat more intense but less homogeneous, with small groups of intensely stained nuclei occasionally being seen in the outer cortex. An approximately 15 to 20-times lower dose of cisplatin than of carboplatin was needed to reach equal staining levels in the liver and kidney. Plateau staining levels in both tissues were reached at between approximately 8 and 48 h after administration of the carboplatin. This was followed by a significant reduction in the kidney samples, whereas the staining levels in the liver section seemed to be more persistent. No major difference was observed between male and female rats in the formation and removal of DNA damage in these tissues. The levels of the various DNA adducts were measured with a competitive ELISA in liver, kidney, spleen, testis, and combined ovary/uterus samples collected at 8 and 48 h after carboplatin administration. At both 8 and 48 h, the highest platination levels were observed in the kidney, followed--in decreasing order--by the liver, combined uterus and ovary samples, spleen, and testis. At 8 h after administration of carboplatin, the relative occurrence of the bifunctional adducts Pt-GG (34%), Pt-AG (27%), and G-Pt-G (32%), was similar in all tissues. The same held for the monoadducts that amounted to about 7% of the total DNA platination. These data indicate that in the first few hours after carboplatin treatment, no preference for the formation of Pt-GG adducts was observed, which confirms our earlier observations obtained with cultured cells. When the total DNA-platination levels (calculated from the sum of the adducts) seen at 8 and 48 h after treatment were compared, a substantial decrease in DNA platination was observed in the kidney (37%), liver (30%) and ovary/uterus (39%), whereas the repair levels in the testis (9%) and, probably, the spleen (18%) were substantially lower. In all tissues studied, only the relative occurrence of the Pt-GG adducts increased between 8 and 48 h, and as a result, at 48 h, after carboplatin administration the Pt-GG adduct was the major adduct persisting in the DNA samples.

Formation of DNA Adducts by the Anticancer Drug Carboplatin: Different Nucleotide Sequence Preferences in Vitro and in Cells

We have studied the formation of adducts upon carboplatin treatment of isolated DNA and in cells. The major adduct formed in vitro, determined with atomic absorption spectroscopy and enzyme-linked immunosorbent assay, was the intrastrand cross-link cis-Pt(NH3)2d(pGpG)(Pt-GG) (58%). cis-Pt-(NH3)2d(pApG) (Pt-AG) (11%), cis-Pt(NH3)2d(GMP)2 (G-Pt-G) (9%), and monofunctionally bound platinum (cis-Pt(NH3)3dGMP (Pt-G), 22%) were formed in smaller amounts. These relative occurrences of the adducts, average values found between 1 and 16 h of incubation, are comparable with those after incubation with cisplatin. The formation of carboplatin-DNA adducts was slow, and about 230-fold more carboplatin than cisplatin (molar dose) was required to obtain equal levels of platination after 4 h of incubation. However, less than 20 times more carboplatin was needed to obtain equal levels of cytotoxicity after 1 h of exposure of CHO cells. The percentages of the carboplatin-DNA adducts after 7-12 h postincubation of the cells (determined with ELISA), Pt-GG (30%), Pt-AG (16%), G-Pt-G (40%), and Pt-G (14%), were different from those of the in vitro data. After 12 h postincubation, the number of interstrand cross-links (determined by alkaline elution) amounted to about 10% of the G-Pt-G adducts and 3-4% of the total amount of adducts. The immunocytochemical detection (with antiserum NKI-A59) of the platinum-DNA modifications showed a pattern similar to that found for the various bifunctional adducts: the initially low levels slowly increased to maximum values within 7-12 h and then slowly decreased. In conclusion, carboplatin forms the same bifunctional adducts as cisplatin.(ABSTRACT TRUNCATED AT 250 WORDS)