Introduction 10 10 15 3 23 6 11 13 24 n n 17 26 Materials and methods Patient characteristics 1 28 n n n n n n 1 n Table 1 Clinical data and EGFR status among primary CRC and their related metastases Clinical data a Case no. Sex Age (years) pTNM Location Neoadjuvant treatment Primary site Synchronous metastases (location number) Metachronous metastases (location number) 1 2 3 4 1 2 3 4 5 1 M 60 T3N1M1 C  + + + + +      2 M 60 T3N2M1 R RCT + + + +       3 M 66 T2N0M1 R  + + + +       4 M 49 T3N2M1 R CT + + +   + + + + + 5 M 70 T3N1M1 C  + + +        6 F 74 T3N1M1 R RCT + + +        7 F 59 T3N2M1 R CT + + +   −     8 M 71 T3N2M1 C  + + +        9 F 55 T3N2M1 R  − + −        10 F 61 T4N1M1 C CT + +    +     11 F 68 T3N1M1 C CT + +    +     12 M 75 T3N1M1 R  + +         13 M 72 T3N1M1 C  + +         14 M 54 T3N0M1 C  + +         15 F 55 T4N1M1 C  + +         16 F 57 T4N2M1 C  + +         17 F 52 T3N1M1 R  + +         18 M 53 T3N0M1 C  + +         19 M 62 T4N2M1 C  + +         20 F 77 T3N2M1 C  + +         21 F 49 T3N1M1 C  + +         22 F 51 T3N2M1 C  + +         23 M 62 T3N2M1 R RT + +         24 F 65 T3N1M1 C CT + +         25 M 56 T4N1M1 C CT + +         26 F 47 T3N1M1 R RT + +         27 F 59 T3N1M1 C  + −         28 F 45 T3N2M1 C  + −         29 F 81 T3N1M1 R  − +         30 M 58 T2N0M1 C  − +         31 M 69 T3N1M1 C  − +         32 F 68 T3N2M1 C  − +         Liver metastases in all cases except for a metachronous pulmonary metastasis and a synchronous ovarian metastasis in patients 7 and 15, respectively C R RCT CT RT a EGFR immunohistochemistry 1 Tissue microarray Tissue blocks appearing to have enough material upon gross inspection were initially selected and hematoxylin–eosin-stained sections were evaluated by two observers (F. Bibeau and F. Boissière-Michot) for the presence of carcinoma. The areas to be used for the construction of the TMAs were marked on the slide and the donor block. Particular attention was made to select invasive cells near the tumor front. The tissues corresponding to selected areas were sampled using a manual arraying instrument (Manual Tissue Arrayer 1, Beecher Instruments, Sun Prairie, WI, USA). Two TMAs were constructed using 0.6-mm tissue cores. The sampling consisted of three malignant cores from different areas of the tumor from a single case of invasive colorectal cancer, placed at specified coordinates. When possible, normal mucosa was also sampled as internal control. After the arraying was completed, TMA blocks were sectioned at a thickness of 4 μm. One section was stained with hematoxylin–eosin, and EGFR immunostaining using the immunohistochemical system kit EGFR pharmDx™ was performed on the adjacent section. Among the 32 cases sampled, 31 primary CRC, 29 metastatic sites, and 28 matched sites were assessable with this technology. EGFR immunohistochemical staining scoring system 6 In addition to these standardized criteria, a semiquantitative analysis was performed taking into account both the staining intensity and the percentage of positive tumor cells. Statistical analysis The cut-off value for negative/positive expression of EGFR was 1% of tumor cells displaying a membranous staining, as specified by the EGFR pharmDx™ scoring guidelines. Data were summarized by frequency and percentage for categorical variables and by means, standard deviations, median, and range for continuous variables. For TMA analysis, the percentage of positive cells was calculated from the mean of triple cores. P Results EGFR expression of primary CRC and their related metastases on tissue sections EGFR reactivity was not homogeneous throughout the tumors. EGFR immunostaining was mainly observed in the deepest region of the primary CRC or at the periphery of metastases, especially in the liver. Of note, EGFR immunoreactivity was often greater in isolated tumor cells and in small clusters of tumor cells. 1 P Nine (28%), 10 (31%), and eight (25%) of the primary CRC and seven (22%), 14 (44%), and nine (28%) of the synchronous metastases displayed a weak, moderate, and strong positivity, respectively. According to the extent of EGFR immunoreactivity, five (16%) primary CRC had no reactivity, 12 (37.5%) had <10% reactive cells, eight (25%) had >10–50% positive cells, and seven (22%) had >50% labeled cells. In the synchronous metastases, two cases (6%) were negative, 13 (41%) had <10% reactive cells, 11 (34%) had >10–50% positive cells, and six (19%) had >50% labeled cells. r 2 P r 2 P 1 1 1 Fig. 1 a b bar  1 1 EGFR expression of primary CRC and their related metastases on TMA 2 Table 2 Comparison of EGFR expression in primary CRC and their synchronous metastatic sites on whole tissue sections and TMA   Primary site Metastases Matched sites Tissue sections 27/32 (84%) 30/32 (94%) 25/32 (78%) First TMA 18/31 (58%) 18/29 (62%) 9/28 (32%) Second TMA 14/31 (45%) 16/29 (55%) 8/28 (29%) Both TMAs 20/31 (65%) 19/29 (66%) 12/28 (43%) Data are expressed as the number of positive cases/total number of  specimens 2 2 Comparison of EGFR expression between tissue sections and TMA P P P P P Discussion HER1 15 15 3 23 4 6 29 6 11 13 24 To answer the question as to whether EGFR expression is similar in different tumor sites, we compared the EGFR status of a set of primary CRC and their corresponding synchronous distant metastases, using a standardized immunohistochemistry test (EGFR pharmDx™, Dako). We also analyzed EGFR expression in metachronous metastases, when available, to study possible changes of EGFR expression during the metastatic process of CRC. 1 P 8 11 13 24 1 1 1 19 22 27 2 3 5 23 24 2 14 16 9 12 20 8 25 2 18 21 30 7