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

Abstract

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.

Keywords: accelerator mass spectrometry; biomarkers of response; diagnostic; microdosing; non-small cell lung cancer; platinum-based chemotherapy; predictive diagnostics.

Figure 1. Illustrations of cisplatin and carboplatin chemical structure and biological mechanisms of action

A. Simplified schematic of cisplatin and carboplatin structure and DNA adduct formation (R=OH or H₂O, * = represents ¹⁴C label, which can be detected by AMS). B. Diagram of the major mechanistic steps of platinum (Pt) based chemotherapy. Formation of Pt-induced DNA damage (adducts) is the most critical step of Pt-induced cell death. Other major steps involved in damage accumulation, such as drug metabolism, cell accumulation (uptake/efflux), intracellular inactivation and DNA can also affect the levels of cytotoxicity.

Figure 2. Linear correlation of IC₅₀ for cisplatin and carboplatin in NSCLC cell lines

Linear correlation of cisplatin IC₅₀ with carboplatin IC₅₀ in A. ten NSCLC cell lines from the NCI-60 panel (R² = 0.66, p = 0.004) and B. six additional NSCLC cell lines used in this manuscript (R² = 0.72, p = 0.033). Only mean values are shown.

Figure 3. Microdose-induced carboplatin–DNA monoadduct levels correlate therapeutic adduct levels

Indicated six NSCLC cells lines were dosed for 4 h followed by washing and incubation in drug-free medium as described. A. Monoadduct formation over time induced by microdoses (1 μM). B. Monoadduct formation over time induced by therapeutic doses (100 μM). C. Dose proportionality of microdose and therapeutic dose induced carboplatin-DNA monoadduct level onto log scale. D. Linear correlation of carboplatin-DNA monoadduct level induced by microdosing and therapeutic carboplatin, showing that the monoadduct levels induced by therapeutic carboplatin was highly linear to the monoadduct levels induced by microdosing carboplatin (R² = 0.95, p < 0.0001). Mean values and standard error are shown.

Figure 4. Correlation between NSCLC cell line sensitivity and carboplatin-DNA monoadduct levels

Box plot (whiskers min to max) comparing monoadduct levels in carboplatin-sensitive (H23, H460, H727) and –resistant (HCC827, H1975, A549) NSCLC cell lines over 24 h. Three most resistant cell lines (white box) had significant (*** = p < 0.001, * = p < 0.05, one-way ANOVA with Bonferroni’s multiple comparison test) lower adduct level A. after microdose or B. therapeutic dose. Comparison of area under the adduct curve (AUC_adduct) between sensitive (grey box) and resistant (white box) in NSCLC cell lines after 4h of treatment with C. 1 μM (microdose) or D. 100 μM (therapeutic dose) of carboplatin. The sensitive cell lines had significant higher AUC_adduct (p < 0.001, student t-test) the resistant NSCLC cell lines. Linear correlation of AUC_adduct E. microdose (R² = 0.70, p = 0.038) or F. therapeutic dose (R² = 0.82, p = 0.014) with cell sensitivity (IC₅₀) towards cisplatin.

Figure 5. No correlation between cisplatin or carboplatin sensitivity and ERCC1 mRNA levels

A. Bar graph of relative ERCC1 (ERCC1/β-actin ratio) mRNA expression level of indicated NSCLC cell lines (grey bar = sensitive, white bar = resistant) shown as mean and SD. Linear regression analysis of B. cisplatin (R² = 0.001, p = 0.950) or C. carboplatin (R² = 0.05, p = 0.686) IC₅₀ with relative ERCC1 expression.

Figure 6. Carboplatin microdose elimination kinetics during a feasibility clinical trial

A. Carboplatin plasma concentration over 24 hours after microdosing as determined from whole plasma by LSC (N = 6 patients). Initial elimination T_1/2α: 38–57 minutes, second phase T_1/2β: 6.7–11.2 hours. B. Pharmacodynamic analysis of carboplatin-DNA monoadducts in PBMC over 24 hours. C. Carboplatin-DNA monoadducts in PBMC (N = 6) and in tumor (N = 3) biopsy specimens at 24h. (red = non-responder, green = responder, black line = no response correlation possible, PR = partial response, SD = stable disease, DP disease progression, NC = no chemotherapy).