Introduction 34 10 17 22 33 1 19 20 18 35 35 18 9 16 30 7 36 26 15 Materials and methods Study population 15 15 21 29 5 Immunohistochemistry and microscopic analysis 5 27 28 31 13 23 31 32 18 3 11 7 Statistical analyses U t 2 P Results Scoring methods 3 11 35 HCA-2 and HC10 staining in rectal cancer 1 1 Table 1 Most rectal tumors have high numbers of tumor cells positive for HCA2 or HC10  N N HCA2  High 324 (65%) 312 (58%)  Low 142 (28%) 174 (32%)  Absence 31 (6.2%) 52 (9.7%) HC10  High 403 (76%) 436 (77%)  Low 117 (22%) 116 (21%)  Absence 8 (1.5%) 12 (2.1%) N Fig. 1 a–c d–f a d  b  e c f  Analysis of HLA class I expression in rectal tumors Together, the results obtained with HCA2 and HC10 are expected to reflect HLA class I expression in rectal cancer. In a group of 64 tumors it was studied whether an additional staining for β2 m would better define HLA class I expression. The results of the addition of β2 m to HCA2 and HC10 were comparable to those obtained with HCA2 and HC10, i.e. only 1 of 64 tumors was differently classified. Therefore, β2 m was not scored in the whole cohort and HLA class I expression was assessed by combining HCA2 and HC10. 2 2    P Table 2 Expression of HLA class I in rectal cancer using HCA2 and HC10 antibodies  HCA2 HC10 HLA class I High (N) N N Irradiated N 270 37 406 (85%) N 99 70 70 (15%) Non-irradiated N 277 32 445 (84%) N 136 87 87 (16%) N 2 P 2 P 2 P HLA class I negative cells and microsatellite instability 8 16 6 4 25 HLA class I expression and clinicopathological parameters 3 P P P Table 3 Clinicopathological characteristics of irradiated and non-irradiated patients with high or low numbers of HLA class I positive tumor cells   Non-irradiated patients Irradiated patients N N P N N P Gender  Male (%) 63 75 0.03 65 66 0.90 Age  Median years 65 68 0.32 65 65 0.99 TNM stage (%)  I 31 24 0.52 33 24 0.01  II 27 30  30 21   III 36 38  32 40   IV 5 8  5 14  Circumferential margin  Negative (%) 83 77 0.28 86 74 0.02 Distant from anal verge (%)  ≥10 cm 28 33 0.17 27 32 0.30  5–10 cm 41 31  46 36   < 5 cm 31 36  27 32  Operation type (%)  Low anterior resection 66 61 0.77 65 66 0.89  Abdomino-perineal resection 29 33  29 30   Hartmann 5 6  6 4  N P Expression of HLA class I and clinical prognosis 15 2 P P P P P 2 4 Fig. 2 a–d a b c d a c b d low P  Table 4 Both irradiated and non-irradiated patients with high expression of HLA class I have a better overall, and disease free survival  Non-irradiated patients Irradiated patients High (%) Low (%) P High (%) Low (%) P Overall survival 65.5 58.5 0.012 67.5 51.3 0.008 Disease free survival 62.2 53.5 0.015 62.2 48.3 0.006 Cancer specific survival 74.3 71.4 0.41 80.1 61.8 0.003 Local recurrence 8.9 13.7 0.22 4.7 3.2 0.72 Distant recurrence 26.7 28.7 0.88 24.7 29.3 0.34 P P Multivariate analysis 5 P P Table 5 Multivariate analysis confirms independent better overall, and disease free survival for rectal cancer patients with high expression of HLA class I  Overall survival Disease free survival Cancer specific survival HR (95% CI)  P HR (95% CI)  P HR (95% CI)  P HLA  High 1 0.042 1 0.006 1 0.653  Low 1.3 (1.0–1.6)  1.4 (1.1–1.8)  1.1 (0.8–1.5)  Randomization  TME 1 0.632 1 0.214 1 0.282  TME + RT 1 (0.8–1.2)  0.9 (0.7–1.1)  1.1 (0.9–1.5)  TNM  I 1  1  1   II 2.2 (1.7–3.0) <0.001 2.1 (1.6–2.8) <0.001 3.5 (2.0–6.1) <0.001  III 3.1 (2.4–4.1) <0.001 3.1 (2.3–4.0) <0.001 9.0 (5.4–14.9) <0.001  IV 11.8 (8.1–17.1) <0.001 – – 50.3 (28.5–89.1) <0.001 CRM  Negative 1 <0.001 1 <0.001 1 <0.001  Positive 1.3 (1.1–1.5)  1.8 (1.4–2.2)  1.3 (1.1–1.5)  HR CI TME RT CRM P P Discussion 18 35 26 Materials and methods 18 35 35 14 24 30 8 16 4 14 25 16 2 16 12 24 Our results show that HLA class I expression in rectal cancer affects the patient’s prognosis. We hypothesize that both oncogenic pathway and HLA class I expression dictate clinical tumor progression. We suggest that in future prognostic studies, analyzing expression of HLA class I or other biomarkers in colorectal cancer, the impact of MSI should be considered.