*4.4. Statistical Analyses*

In the evaluation phase, DFS was calculated with respect to the groups of patients divided by the genotype (common homozygous, heterozygous, and rare homozygous). The log-rank test for each variant was performed and the Kaplan–Meier plot was generated for visual inspection of gene dosage. We set the study follow-up end to 120 months (10 years) and thus, all subjects with DFS exceeding 120 months were censored. The response of patients to NACT was set to "good" in the case of complete or partial pathological remission (CR/PR) and "poor" for stable or progressive disease (SD/PD). We evaluated associations between genotypes (common homozygous, heterozygous, and rare homozygous) and response using the Pearson chi-square test. Adjusted *p*-value was calculated for each variant and each of these tests. Adjusted *p*-value for the log-rank test was based on 100 permutations of original data. A *p*-value of less than 0.05 after adjustment for multiple testing was considered statistically significant. Variants significantly associating with either DFS or response to NACT in the evaluation phase entered the validation phase of the study.

In the validation phase, the Pearson chi-square test and the log-rank tests were used as described above. For the evaluation of allele effect, recessive, dominant, co-dominant, and additive genetic models were used. Association of variants with transcript levels was assessed by the non-parametric Kruskal–Wallis test. Adjusted *p*-values were calculated using Benjamini–Hochberg false discovery rate (the FDR test) as a correction for multiple testing [47]. Haplotype analysis was conducted in HaploView 4.2 (Broad Institute, Cambridge, MA, USA). Statistical analyses were conducted using R and the statistical program SPSS v16.0 (SPSS, Chicago, IL, USA).

The sequencing data that support the findings of this study are openly available in Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra) under accession no. PRJNA510917.

**Supplementary Materials:** Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/24/ 9556/s1.

**Author Contributions:** Conceptualization, P.S., V.H., and R.V.; methodology, V.H.; software, P.S. and V.H.; validation, P.S., V.B., and V.H.; resources, R.K., K.K., D.V., and J.G.; data curation, R.K., K.K., D.V., and J.G.; writing—original draft preparation, P.S. and V.H.; writing—review and editing, all authors; visualization, P.S., and V.H.; supervision, P.S.; project administration, P.S., V.H., and D.V.; funding acquisition, P.S., V.H., and V.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Czech Medical Council, grant number 17-28470A to P.S., the Czech Ministry of Education, Youth and Sports, grant number CZ.02.1.01/0.0/0.0/16\_013/0001634 to V.H., the Grant Agency of Charles University, grant number UNCE/MED/006 to V.B. and the Grant Agency of the Czech Republic, grant number 19-03063S to P.S.

**Acknowledgments:** Authors would like to thank Pavel Ostašov from Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Czech Republic for help with computing in R software environment and all participating patients for their kind consent to the study and clinical personnel for outstanding support.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
