Introduction 1 2 11 13 21 28 5 11 21 Since a substantial number of patients treated with concurrent chemoradiation still fail to respond to this toxic treatment, there is a need for an accurate predictive assay, based on which patients that are likely to respond to the therapy can be selected. This strategy may also provide a tool to individualize and tailor treatment, based on evaluation of the predictive assay, early during therapy. 8 34 3 17 32 35 3 32 35 In our institute, study protocols with cisplatin-DNA adduct measurements are ongoing in patients with HNSCC and cervical cancer, who are all treated with concurrent cisplatin-based chemoradiation. The objectives of the current study are (1) to investigate the two major forms of cisplatin-DNA adducts (GG and AG adducts) after different schedules of cisplatin given concurrently with radiation and (2) to explore relationships between adducts in primary tumor and normal tissue. We specifically wanted to investigate whether the level of adducts in tumors are reflected by those in normal tissues. In studies focused on the predictive value of cisplatin-DNA adduct levels, this would then justify the use of more easily obtained normal tissues as a surrogate for tumor samples. Patients and methods Concurrent chemoradiation protocols This study on adduct formation was approved by the medical ethical committee of the participating hospitals. The main eligibility criteria were: patients scheduled for cisplatin chemoradiation, no previous treatment with cisplatin, and informed consent. Eligible patients were informed about the nature of the protocol and after written informed consent they were entered in the study. Patients were recruited from one of the following regimens. RADPLAT 100 11 2 RADPLAT daily LD 2 6 19 CERVIX 40 2 Cisplatin-DNA adducts 24 32 29 29 Statistical analysis t Results Samples for cisplatin-DNA adduct determination were obtained from 63 patients: 27 from RADPLAT daily LD, 15 from CERVIX 40, and 21 from RADPLAT 100. WBC samples were taken from 61 patients, buccal cells from 25, and tumor biopsies from 23 of these patients. The reasons for the missing data for the normal tissue samples were: no collection of samples due to logistics or (in minority of cases) not sufficient volume for analysis. The reason for missing primary tumor biopsy data were: tumor not accessible for direct outpatient-based biopsy (in HNSCC patients) or refusal (in cervix cancer patients). In WBC, all but three of the 60 available baseline samples were below the LLQ for the GG adducts and all but two below the LLQ for the AG adducts. This was probably due to some background signal inherent in the postlabeling method, since all patients had not been treated before with platinum chemotherapy. The yield of DNA, obtained from the buccal cell samples was rather low, ranging from 1–10 μg. Baseline samples of buccal cells were available from 16 of 25 patients, of whom posttreatment samples were also available. The baseline values of GG adducts in buccal cells ranged from 0.067 to 0.745 fmol/μg DNA (mean 0.282, SD 0.19) and baseline values of AG adducts in buccal the cells ranged from 0.087 to 1.538 fmol/μg DNA (mean 0.398, SD 0.38). All but two of the baseline GG-adduct values were above the LLQ and all the baseline AG-adduct values were above the LLQ. This was probably due to the low DNA quantities obtained from the buccal cell samples. The difference in adduct levels from baseline to post-infusion values was significant for the GG adducts, but not for the AG adducts. This implies that for measuring low quantities of AG adducts in the low amounts of buccal cell DNA available, we reached the limits of quantification with this postlabeling method. 1 t P t P t P Table 1 Cisplatin-DNA adducts (in fmol/μg DNA) in normal tissue and primary tumor after different schedules of cisplatin-based chemoradiation Treatment schedule WBC Buccal cells Tumor GG AG GG AG GG AG RADPLAT daily LD N 26 26 11 11 6 6  Mean 0.34 0.05 0.84 0.21 0.66 0.10  SD 0.10 0.19 0.39 0.14 0.37 0.05 CERVIX 40 N 14 14 7 7 10 10  Mean 0.44 0.06 0.87 0.22 1.94 0.26  SD 0.17 0.03 0.26 0.06 1.47 0.26 RADPLAT 100 N 21 21 7 7 7 7  Mean 1.047 0.124 1.563 0.340 3.866 0.413  SD 0.377 0.049 0.434 0.090 1.101 0.089 See text for explanation of treatment schedules N SD WBC GG AG r P n 1 r P n 1 r P n 1 Fig. 1 a b c red squares green circles blue triangles  r P n r P n r P n 2 r P n r P n r P n Fig. 2 a b c red squares green circles blue triangles  Discussion There are two main conclusions from the 63 patients included in these analyses. First, intra-tumoral adduct levels were substantially higher than those in normal tissue (WBC) at all cisplatin-dose levels examined. Second, no positive correlations were evident between adducts in tumors and normal tissues. It should be noted that the various schedules, the cisplatin doses, and the duration of infusions differed, as well as the sampling times. However, all analyses on adducts were performed on paired samples, within the same patient. This eliminates variance of these factors since the normal tissue and tumor samples all were obtained at similar time points after the cisplatin infusion. In the RADPLAT daily LD, some accumulation from the previous four daily 6 mg cisplatin infusions would have occurred and affected the day 5 measurement after the 5th infusion. In the RADPLAT 100 and CERVIX 40 patients, no such accumulation would have occurred, since the sampling was done 20–23 h after the first infusion of cisplatin. 17 23 12 33 15 22 37 23 9 30 18 33 1 8 36 7 36 25 22 20 32 36 24 The results from the buccal cell samples need to be interpreted with caution, especially the AG-adduct levels, since uncertainties remain. With the low quantities of AG-adducts in the low amounts of buccal cell DNA we could extract, we reached the limits of quantification of the postlabeling method. 3 4 10 14 26 31 32 35 31 32 4 26 14 10 3 35 17 1 27 16 In conclusion, we have demonstrated that in concurrent chemoradiotherapy schedules, cisplatin adduct levels in tumors were significantly higher than in normal tissues (WBC). No evidence of a correlation was found between adduct levels in normal tissues and primary tumor biopsies. This lack of correlation may, to some extent, explain the inconsistencies in the literature regarding whether or not cisplatin-DNA adducts can be used as predictive test in anticancer therapy. Conflict of interest: none declared.