Introduction 1 2 5 2 2 6 7 2 8 10 11 14 15 16 17 18 18 20 21 24 25 26 Materials and methods Clinical characteristics and tissue samples From 109 patients with cervical carcinoma who underwent radical hysterectomy with bilateral pelvic lymphadenectomy (by the same surgical team) between 1985 and 1999, formalin-fixed, paraffin-embedded tissue blocks were retrieved from the archives of the Department of Pathology, Leiden University Medical Centre, The Netherlands. Twenty-six adenocarcinomas and 83 squamous carcinomas were obtained. These cases were not consecutive, but chosen on the basis of availability of sufficient material. The use of clinical material was approved by the institutional review board according to the guidelines of the Dutch Federation of Medical Research Associations. All patients were inhabitants of The Netherlands and had not received pre-operative radiotherapy or chemotherapy. Mean age was 48.5 years, the youngest patient being 24 years and the oldest 87 years at the time of surgery. Follow-up of these patients until 2005 provided information concerning recurrence rate and performance state. Tissue microarray construction The archival slides for all the cases were reviewed; a slide containing representative tumour was selected, and an area of tumour was encircled on the slide. The corresponding tissue blocks were recovered from the archives and the selected area on the slide was circled on the block for tissue microarray construction. Using a manual tissue microarrayer (Beecher Instruments, Silver Spring, MD, USA), the area of interest in the donor block was cored with a 0.6-mm diameter needle and transferred to a recipient paraffin block. The microarray was constructed with a threefold redundancy (three spots for each patient) to increase accuracy. The finalized arrays were then cut into 3 μm-thick sections and mounted on glass slides using an adhesive tape-transfer system (Instrumedics Inc., Hackensack, NJ, USA) with ultraviolet cross-linking. Antibodies 2 27 28 2 2 27 29 30 30 30 31 31 32 32 32 32 2 33 35 Immunohistochemical staining 36 Evaluation of TMA immunostaining 37 Statistical analysis The Chi-square test or, where appropriate, Fisher’s exact test was used for evaluation of associations between expression and clinicopathological parameters. All statistical analyses were performed with the SPSS Version 12 software package. Five-year survival rates were calculated and survival rates were compared according to the Kaplan–Meier method using the log rank test, while multivariate analysis of survival was performed according to Cox proportional hazard models. Overall survival (OS) was defined as survival till death due to cervical carcinoma (the two patients that dyed from other causes were excluded), i.e. disease-specific survival, while disease-free survival (DFS) was defined as time to disease recurrence, metastasis, or disease-specific death. All tests were two-sided and the significance level was set to 5%. Results Clinical features Of the total group of 109 patients, 52 were diagnosed as FIGO stage IB1, 30 as IB2, 21 as FIGO IIa and 2 as FIGO IIb. For four patients no FIGO staging was available. Fifty-five patients (50%) received post-operative radiotherapy due to narrow tumour-free margins of excision, lymph node metastases, vaso-invasive growth, parametrial infiltration, depth of infiltration exceeding 15 mm and/or tumour size exceeding 4 cm. Twenty-seven patients had lymph node metastases. Fifty-one patients (58%) were HPV16-positive, 21 (24%) were HPV18-positive, while the remaining patients were positive for HPV31, HPV33, HPV45, HPV52 or HPV68. At the end of follow-up, 80 patients were alive, 2 suffered from recurrent disease, 3 had a metastasis, 22 had died of the disease and 2 had died of causes unrelated to the primary disease. The median follow-up time was 45 months. HLA class I and APM component expression 1 2 2 1 2 2 2 Fig. 1 a–c d–f g–i  j–l  a, d, g, j  b, e, h, k  c, f, i, l   Fig. 2 a b   Table 1 2  HLA-A HLA-B/-C 2 HLA class I Total loss Partial loss Normal expression Total loss Partial loss Normal expression Total loss Partial loss Normal expression Total loss Partial loss Normal expression n 36 (33) 22 (20) 51 (47) 24 (22) 29 (27) 56 (51) 8 (7) 28 (26) 73 (67) 21 (19) 24 (22) 63 (59) Tumour type n 6 (23) 5 (19) 15 (58) 4 (15) 8 (31) 14 (54) 1 (4) 7 (27) 18 (69) 4 (15) 5 (19) 17 (66) n 30 (36) 17 (21) 36 (43) 20 (24) 21 (25) 42 (51) 7 (8) 21 (25) 55 (67) 17 (21) 19 (23) 46 (56) n 2 P P Table 2 Overall and histological type-specific expression of APM components  n n n Total loss Partial loss Normal expression P Total loss Partial loss Normal expression P Total loss Partial loss Normal expression P LMP2 14 (14) 30 (30) 57 (56) 0.014 5 (20) 11 (44) 9 (36) 0.772 9 (12) 19 (25) 48 (63) 0.001 LMP7 3 (3) 16 (16) 82 (81) <0.001 2 (8) 9 (36) 14 (56) 0.100 1 (1) 7 (9) 68 (90) <0.001 LMP10 0 0 101 (100)          TAP1 4 (4) 19 (19) 78 (77) <0.001 0 (0) 4 (16) 21 (84) 0.230 4 (5) 15 (20) 57 (75) 0.001 TAP2 11 (11) 26 (26) 64 (63) <0.001 4 (16) 6 (24) 15 (60) 0.005 7 (9) 20 (26) 49 (65) <0.001 ERAP1 0 14 (15) 80 (85) 0.314 0 (0) 2 (9) 20 (91) 0.582 0 12 (16) 60 (84) 0.307 Tapasin 1 (1) 24 (24) 76 (75) <0.001 0 (0) 10 (40) 15 (60) 0.284 1 (1) 14 (18) 61 (81) <0.001 Calnexin 0 0 101 (100)          ERp57 12 (12) 40 (40) 49 (48) 0.017 1 (4) 6 (24) 18 (72) 0.615 11 (15) 34 (45) 31 (40) 0.002 Calreticulin 0 0 101 (100)           n  P Association with histopathological parameters P P Association with overall and disease-free survival 3 3 4 Table 3 HLA class I and APM component expression and 5-years survival rates  Overall survival Disease-free survival Normal expression Partial loss Total loss Normal expression Partial loss Total loss HLA-A 42 (83) 12 (55) 29 (81) 42 (83) 13 (60) 28 (77) HLA-B/-C 45 (81) 19 (64) 18 (75) 46 (82) 19 (67) 18 (73) HLA class I 52 (83) 15 (61) 16 (77) 52 (82) 16 (65) 15 (75) LMP7 24 (74) 17 (87) 1 (33) 62 (76) 13 (80) 2 (50) TAP1 61 (78) 8 (70) 1 (33) 63 (81) 13 (67) 1 (25) ERAP1 66 (82) 5 (38) – 65 (81) 7 (50) –  n Fig. 3 a b  c  d  e  f P  Fig. 4 a  b  c  d  e  f  P  Decreased OS and DFS was significantly associated with total TAP1 loss, but not with normal expression and partial loss. A similar pattern was observed for partial LMP7 loss, though this was not significantly associated with DFS. ERAP1 loss was significantly associated with decreased OS and DFS as compared to normal ERAP1 expression. Multivariate analysis 4 P P P P P Table 4 Histopathological parameters and 5-years survival rates  Overall survival Disease-free survival % P % P Depth of infiltration n 88  90  n 60 <0.001 57 <0.001 Vaso-invasion n 81  85  n 59 0.031 54 0.002 Lymph nodes n 86  86  n 43 <0.001 39 <0.001 Histopathological class n 75  76  n 77 0.924 74 0.662 FIGO stage n 82  91  n 69 0.060 57 0.002 Discussion In the present study we have investigated HLA class I and APM component expression in cervical carcinoma patients and have evaluated the relationship of these factors with survival and other clinicopathological parameters. 19 21 9 18 20 30 33 37 2 2 14 34 38 41 41 42 43 45 Although we found that total TAP1 loss was an independent predictor for shorter DFS, this finding should be interpreted with caution as only four patients exhibited this loss. The decreased significance of ERAP1 as an independent predictor for shorter DFS can be explained by the interdependency of ERAP1-mediated peptide trimming and TAP-mediated peptide transport. 46 47 48 49 2 2 50 52 8 In conclusion, we have shown here that defective expression of HLA class I and of various APM components occurs frequently in cervical carcinoma. Partial HLA class I loss and total TAP1 loss are associated with decreased survival, while down-regulation of immunoproteasomal subunits is associated with decreased risk of lymphogenic metastasis. Moreover, we report the first description of ERAP1 down-regulation in a human carcinoma, and have found that this is an independent prognostic parameter for decreased survival. A complete understanding of the mechanisms and relevance of HLA class I and APM component down-regulation and immune evasion may contribute to the rational design of tumour vaccines and T-cell-based immunotherapies.