After subdividing the patients by administered drugs/major drug subgroups, as previously mentioned, a total of 17 GWAS analyses were carried out by comparing the allele/genotype frequency between the patients who had developed severe neutropenia/leucopenia (grade 3/4) to those who had not developed any adverse drug reactions. The Q–Q plots of each GWAS and the calculated lambda value of below 1.00 indicated no significant population stratification in each of these GWAS analyses (Fig. S1). From this study, although we could not identify any SNPs that surpassed the genome-wide significant threshold (P-value < 5 × 10−8) for showing association with the risk of neutropenia/leucopenia induced by the certain type of drug or regimen, several possible candidate loci were identified. The results of the GWAS are summarized in Table 2, Table S1, and Figure S2; the results of wGRS are summarized in Table S2.
Among these datasets, GWAS carried out using samples who were given: (i) any kind of platinum-based chemotherapy (428 cases vs 743 controls); (ii) cisplatin-based chemotherapy (176 cases vs 471 controls); or (iii) carboplatin-based chemotherapy (261 cases vs 262 controls) identified SNPs showing the most significant association with chemotherapy-induced severe neutropenia/leucopenia are: rs4886670 (Pmin = 9.86 × 10−7, OR = 1.61, 95% CI = 1.33–1.94) near RPL36AP45 for (i); rs10253216 (Pmin = 1.68 × 10−7, OR = 1.48, 95% CI = 1.16–1.89) near AGR2 for (ii); and rs11071200 (Pmin = 8.51 × 10−7, OR = 8.24, 95% CI = 2.89–23.5) on PRTG for (iii) (Table 2, Table S1, Fig. S2b). For the anthracycline-based regimen, we carried out GWAS with individuals given all anthracycline-based (184 cases vs 459 controls), doxorubicin-based (83 cases vs 66 controls), and epirubicin-based (83 cases vs 370 controls) chemotherapy, and identified three SNPs, rs10040979 (Pmin = 4.60 × 10−7, OR = 1.45, 95% CI = 1.12–1.88) in EBF1, rs11857176 (Pmin = 8.08 × 10−7, OR = 1.80, 95% CI = 1.13–2.87) near a hypothetical gene LOC100302666, and rs4149639 (Pmin = 2.89 × 10−7, OR = 4.44, 95% CI = 2.57–7.68) in TNFRSF1A, to be most significantly associated with the risk of high-grade neutropenia/leucopenia, respectively (Table 2, Table S1, Fig. S2c). In the case of antimicrotubule agents, we carried out three different GWAS with individuals who were treated with antimicrotubule (371 cases vs 825 controls), paclitaxel-based (218 cases vs 364 controls), or docetaxel-based (147 cases vs 233 controls) regimens. We identified three SNPs, rs11651483 (Pmin = 3.37 × 10−7, OR = 1.36, 95% CI = 1.12–1.64) in RICH2, rs922106 (Pmin = 9.28 × 10−7, OR = 1.68, 95% CI = 1.28–2.21) in LRRC8B and rs3747851 (Pmin = 5.61 × 10−7, OR = 2.38, 95% CI = 1.69–3.34) in DAB2IP, to be those most significantly associated with the increased risk of severe neutropenia/leucopenia, respectively (Table 2, Table S1, Fig. S2e). Our previous report by Kiyotani et al. identified four SNPs to be associated with gemcitabine-induced hematological toxicities. Three of the four SNPs were included in the current study with suggestive association, rs12046844 (Pmin = 5.84 × 10−4, OR = 2.53, 95% CI = 1.45–4.43), rs6430443 (Pmin = 8.61 × 10−4, OR = 6.33, 95% CI = 1.90–22.2; r2 = 0.895 with rs1901440) and rs11719165 (Pmin = 1.16 × 10−2, OR = 2.36, 95% CI = 1.18–4.70) (Table S4). However, it is noted that some of the samples used in this study overlapped with those in the study reported by Kiyotani et al., as both sourced samples from Biobank Japan.
Lastly, we also attempted to identify genetic variants associated with combined treatment of paclitaxel and carboplatin-induced severe neutropenia/leucopenia (150 cases vs 166 controls), as this combined treatment is commonly used as the standard therapy for both ovarian and lung cancers. We found the most significant association with the SNP rs12310399 (Pmin = 2.46 × 10−7, OR = 1.85, 95% CI = 1.33–2.58) near the FGD6 gene (Table 2, Table S1, Fig. S2a), which is suggested to activate CDC42, a member of the Ras-like family of Rho and Rac proteins, and has a critical role in regulating the actin cytoskeleton. The second strongest association was observed at the locus encoding RXRA (Pmin = 7.38 × 10−7, OR = 2.58, 95% CI = 1.77–3.77), an important transcriptional factor. We also calculated the cumulative genetic scores using SNPs on six loci and identified that individuals in group 4 could have 188 times (95% CI = 36.1–979) higher risk of developing severe neutropenia/leucopenia than those belonging to group 1 with the sensitivity of 95.9% and the specificity of 88.9% (Table S2). Because this drug combination is of clinical importance, we further investigated the association of these six selected loci using 161 individuals who developed grade 1/2 neutropenia/leucopenia, using cases registered in the Biobank Japan. Interestingly, the association results for the six loci were moderate for grade 1/2 neutropenia/leucopenia, with intermediate allele frequency and OR between individuals without any adverse reactions and those with neutropenia/leucopenia of ≥grade 3 (Table S3). In addition, as shown in Table 3 and Figure 1, the higher the calculated score becomes, the higher the proportion and grade of neutropenia/leucopenia. The intermediate scores for patients with grade 1/2 neutropenia/leucopenia could imply the possible usefulness of this scoring system for the prediction.
Figure 1. Proportions of cancer patients who developed no adverse reaction (G0), mild neutropenia/leucopenia (G1/2), or severe neutropenia/leucopenia (G3/4) in each of the weighted genetic risk score (wGRS) score groups. All patients received combined treatment with paclitaxel and carboplatin and were registered with Biobank Japan. The total numbers of patients in scores 1, 2, 3, and 4 are 71, 171, 159, and 73, respectively.
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Furthermore, we used simulation to estimate how many samples are required to validate this scoring result. We started off by estimating the incidence of neutropenia/leucopenia by the combined treatment of paclitaxel and carboplatin. In Biobank Japan, a total of 477 individuals received this combined treatment; among them, 166 individuals (35%) did not develop any adverse drug reactions, 161 (35%) developed mild neutropenia/leucopenia (grade 1 or 2) and 150 (30%) developed severe neutropenia/leucopenia (grade 3 or higher). The frequency of developing severe neutropenia/leucopenia is in agreement with a multicenter study reported by Guastalla et al. When we assume that 100 patients who receive this combination therapy are prospectively registered, the incidences of the adverse drug reactions are estimated as shown in Table 4. If we categorize the patients by wGRS according to the proportions indicated in Table 3 (and our hypothesis is right), the statistical power should be enough to validate by this small subset of patients. Even if two individuals in both group 1 and group 4 are incorrectly predicted, the calculated P-value is still 0.03 by Fisher's exact test.