All statistical analyses were carried out in SPSS Statistics for Windows 20.0 (Chicago, IL, USA).
2.9.1 ATM
Differences in ATM expression between tissue types were analysed using paired-sample t- tests. ATM expression was compared with MMR protein expression and clinicohistopathological data using Pearson χ2 and Fisher's exact test. Survival analyses were
performed separately in patients who received preoperative radiotherapy and those who did not. Disease-free and overall survival data were analysed using the Kaplan-Meier method and a log-rank test. Univariate and multivariate analyses were performed using Cox proportional hazards survival modelling for ATM expression in the TC and TP. Covariates included sex, age, TNM stage, tumour grade, vascular invasion, perineural invasion, treatment with chemotherapy and radiotherapy, and TRG. P<0.05 was considered statistically significant. 2.9.2 MRE11
Paired t-tests were used to compare weighted scores between the low and high expressing MRE11 groups in the TC and TP. The scores between the tumour and healthy tissue were also analysed in this way. The Fisher’s exact test was used to test for associations between MRE11 status and the available clinicopathologic variables for our cohort. For these tests, the significance threshold was set to P< 0.05.
Furthermore, Cox regression models were used to test for univariate associations between the clinicopathological variables according to MRE11 status in the TC and TP. The assumption of proportional hazards was tested using log minus log plots; where variables met the assumption of proportional hazards if the plot showed a parallel curve between the groups over time. An interaction term with time was created for variables that did not meet the proportional hazards criteria and that interaction term was added to the model (grade did not meet the criteria for proportional hazards). If a variable was significant in one or both MRE11 groups for TC and TP in the univariate analysis, it was included in the multivariate analysis. Variables were excluded until only significant variables remained in one or both of the MRE11 groups. Two- way interactions between the remaining variables were explored and included in the models if they were significant. Subgroup analysis was performed on lymph node-positive subjects and adenoma-positive subjects to determine whether there was a difference in survival between these groups according to MRE11 high or low status.
2.9.3 ATM/MRE11 combinatorial panel
ATM expression was compared with clinicohistopathological data using Pearson’s χ2 test, and the association of MRE11 expression with clinicohistopathological variables was assessed by Fisher’s exact test. ATM and MRE11 expression levels were compared and combined by binary logistic regression, as described previously. Survival analyses were performed in the overall cohort and separately in patients who received preoperative radiotherapy. Univariate and multivariate analyses were performed using Kaplan Meier curves and Cox’s proportional hazards survival modeling for the combined two-marker expression levels from cancer core and periphery samples. Covariates were sex, age, TNM
stage, grade, vascular invasion, perineural invasion, treatment with chemotherapy and radiotherapy, and TRG. Univariate analysis by the Mann–Whitney U test was also used to assess associations between the single and combined two-marker expression levels in rectal tumour tissue with TRG, which was further characterized with receiver operating characteristic—area under curve (ROC-AUC) analysis. P<0.05 was considered statistically significant.
2.9.4 NSB1
Survival analyses were conducted both for the entire cohort, and separately, in samples from patients who received pre-operative radiotherapy, as has been described previously. In addition, further subgroup analysis was conducted with early tumour stage and low-grade tumours as covariates. Univariate and multivariate analyses were performed using Kaplan– Meier curves and Cox’s proportional hazards survival modelling for NBS1 protein expression in the TC and TP. The covariates included were sex, age, TNM stage, tumour grade, vascular invasion, perineural invasion, chemotherapy and radiotherapy, and TRG. Univariate analysis was performed using the Mann–Whitney U test. P < 0.05 was considered statistically significant.
2.9.5 RAD50
Survival analyses were conducted both for the entire cohort, and separately, in samples from patients who received pre-operative radiotherapy. In addition, further subgroup analysis was conducted with early tumour stage and low-grade tumours as covariates. Univariate and multivariate analyses were performed using Kaplan–Meier curves and Cox’s proportional hazards survival modelling for RAD50 protein expression in the TC and TP. The covariates included were sex, age, TNM stage, tumour grade, vascular invasion, perineural invasion, chemotherapy and radiotherapy, and TRG. Univariate analysis was performed using the Mann– Whitney U test. P < 0.05 was considered statistically significant.
2.9.6 MRE11, NBS1, RAD50 combinatorial panel
Statistical analysis was performed with SPSS Statistics for Windows 20.0 (Chicago, IL, USA). Survival analysis was conducted both for the entire cohort and, separately, in patients who received preoperative radiotherapy. MRE11, RAD50, and NBS1 expression were compared and combined by binary logistic regression (detailed raw data available upon request). Univariate and multivariate analyses of the combined expression of the three proteins at the TC and TP were performed using Kaplan–Meier curves and Cox’s proportional hazards survival modelling. Covariates were sex, age, TNM stage, histological grade, vascular invasion, perineural invasion, chemotherapy, and radiotherapy. Univariate analysis was performed using the Mann–Whitney U test. P<0.05 was considered statistically significant.
2.10 References
Angèle, S., Falconer, A., Foster, C.S., Taniere, P., Eeles, R.A., and Hall, J. (2004). ATM Protein Overexpression in Prostate Tumors: Possible Role in Telomere Maintenance. Am. J. Clin. Pathol. 121, 231–236.
Baker, M. (2016). 1,500 scientists lift the lid on reproducibility. Nature 533, 452–454.
Cardano, M., Diaferia, G.R., Falavigna, M., Spinelli, C.C., Sessa, F., DeBlasio, P., and Biunno, I. (2013). Cell and tissue microarray technologies for protein and nucleic acid expression profiling. J. Histochem. Cytochem. 61, 116–124.
Edge, S.B., and Compton, C.C. (2010). The American Joint Committee on Cancer: the 7th Edition of the AJCC Cancer Staging Manual and the Future of TNM. Ann. Surg. Oncol. 17, 1471–1474.
Edwards, S.J.L., Stone, T., and Swift, T. (2007). Differences between research ethics committees. Int. J. Technol. Assess. Health Care 23, 17–23.
Emanuel, E.J., Wendler, D., and Grady, C. (2000). What makes clinical research ethical? JAMA 283, 2701–2711.
Hari, D.M., Leung, A.M., Lee, J.H., Sim, M.S., Vuong, B., Chiu, C.G., and Bilchik, A.J. (2013). AJCC cancer staging manual 7th edition criteria for colon cancer: Do the complex modifications improve prognostic assessment? J. Am. Coll. Surg. 217, 181–190.
Ho, V., Chung, L., Revoltar, M., Lim, S.H., Tut, T.G., Abubakar, A., Henderson, C.J., Chua, W., Ng, W., Lee, M., et al. (2016). MRE11 and ATM expression levels predict rectal cancer survival and their association with radiotherapy response. PLoS One 11, e0167675.
Ho, V., Chung, L., Singh, A., Lea, V., Revoltar, M., Lim, SH., Tut, TG., Ng, W., Lee, M., de Souza, P., Shin, J and Lee, CS. (2017). Early postoperative low expression of RAD50 in rectal cancer patients associates with disease free survival. Cancers. 9, 163.
Ho, V., Chung, L., Singh, A., Lea, V., Abubakar, A., Lim, SH., Ng, W., Lee, M., de Souza, P., Shin, J and Lee, CS. (2018). Overexpression of the MRE11-RAD50-NBS1 (MRN) complex in rectal cancer correlates with poor response to neoadjuvant radiotherapy and prognosis. BMC Cancer. 18, 869.
Holck, S., Nielsen, H.J., Pedersen, N., and Larsson, L.-I. (2015). Phospho-ERK1/2 levels in cancer cell nuclei predict responsiveness to radiochemotherapy of rectal adenocarcinoma. Oncotarget 6, 34321–34328.
Horvath, L., and Henshall, S. (2001). The application of tissue microarrays to cancer research. Pathology. 33, 125–129.
Jamrozik, K. (2004). Research ethics paperwork: what is the plot we seem to have lost? BMJ
329, 286–287.
Kononen, J., Bubendorf, L., Kallioniemi, A., et al. (1998). Tissue microarrays for high- throughput molecular profiling of tumor specimens. Nat Med. 4, 844–847.
Neill, U.S. (2008). Publish of perish, but at what cost? J. Clin. Invest. 118, 2368.
Rodel, F., Keppner, S., Capalbo, G., Bashary, R., Kaufmann, M., Rodel, C., Strebhardt, K., and Spankuch, B. (2010). Polo-like kinase 1 as predictive marker and therapeutic target for radiotherapy in rectal cancer. Am J Pathol 177, 918–929.
Ryan, R., Gibbons, D., Hyland, J.M.P., Treanor, D., White, A., Mulcahy, H.E., O’Donoghue, D.P., Moriarty, M., Fennelly, D., and Sheahan, K. (2005). Pathological response following
long-course neoadjuvant chemoradiotherapy for locally advanced rectal cancer. Histopathology 47, 141–146.
Tut, T.G., Lim, S.H.S., Dissanayake, I.U., Descallar, J., Chua, W., Ng, W., de Souza, P., Shin, J.-S., and Lee, C.S. (2015). Upregulated Polo-Like Kinase 1 Expression Correlates with Inferior Survival Outcomes in Rectal Cancer. PLoS One 10, e0129313.