Introduction 13 13 1 20 16 24 26 32 21 14 2 4 6 7 9 12 23 35 38 39 41 The aim of the present study was, therefore, to validate TMA technology in ESCC by assessing the concurrence of immunohistochemical staining scores of established molecular markers with various expression patterns between triplicate 0.6 mm core biopsies of the TMA and their whole tissue section counterparts. Materials and methods TMA construction Formalin-fixed, paraffin-embedded tissues from thoracic ESCCs of consecutive patients having undergone esophagolymphadenectomy at the authors’ institute between 1989 and 2006 were retrieved from the archives of the Department of Pathology. Patients who received neoadjuvant therapy were excluded from this study. The study was carried out in accordance with the ethical guidelines of our institution concerning informed consent about the use of patient’s materials after surgical procedures. By an experienced pathologist (FtK), three representative tumor regions were marked on one selected hematoxylin and eosin (H&E)-stained section of each tumor, avoiding areas of necrosis. From these three tumor regions, a tissue cylinder with a diameter of 0.6 mm was punched out of the corresponding paraffin block (‘donor block’) and placed into the TMA paraffin block using a manual tissue arrayer (MTA-I, Beecher Instruments, Sun Prairie, USA), which was guided by the MTABooster® (Alphelys, Plaisir, France). The distribution and position of the cores was determined in advance with the TMA-designer Software (Alphelys-TMA Designer®, Version 1.6.8, Plaisir, France). Cores of normal esophageal mucosa, lymph node, kidney, liver, spleen, and prostate were incorporated in the tissue array block as internal controls. Immunohistochemistry 1 Table 1 Specification of antibodies used and details of tissue processing Primary antibody Staining pattern a Clone and code Antigen retrieval Dilution Incubation time (min/room temperature) b Positive control Procedure CK5/6 Cytoplasmic Chemicon D5/16 B4 EDTA pH 9.0 1:3,000 60 Strept ABC Breast Autostainer CK14 Cytoplasmic Neomarkers LL002 EDTA pH 9.0 1:400 60 Powervision Breast Autostainer E-cadherin Membranous Zymed 4A2C7 Citrate autoclave pH 6.0 1:200 60 Powervision Breast Autostainer MIB-1 (Ki-67) Nuclear Dako M7240 Citrate pH 6.0 1:100 60 Strept ABC Tonsil Autostainer p53 Nuclear Biogenex BP53-12 Citrate pH 6.0 1:200 60 Strept ABC Serous adenocarcinoma of the endometrium Autostainer a b 1 Immunohistochemical scoring 37 10 40 29 30 11 Statistical analysis Statistical analyses were performed using the SPSS software for Windows (Version 12.0, SPSS, Chicago, IL, USA). Sixty-four donor blocks (60% of the tumors incorporated in the TMA) were randomly chosen by means of a random selection function of SPSS. κ κ κ κ κ κ 17 15 Results 2 Table 2 Overview of the amount of cores that were evaluable, absent or contained too little tumor in all 108 cases and in the 64 randomly selected cases on the TMA slides   H&E CK5/6 CK14 E-cadherin Ki-67 p53 Median n  No. of evaluable cores 293 309 294 306 293 295 295  Percentage 90 95 91 94 90 91 91  No. of absent cores 20 7 22 9 22 22 21  Percentage 6 2 7 3 7 7 6  No. of cores without tumor 11 8 8 9 9 7 9  Percentage 4 3 3 3 3 2 3 n  No. of evaluable cores 176 187 176 185 176 176 176  Percentage 92 97 92 96 92 92 92  No. of absent cores 13 3 13 4 13 13 13  Percentage 7 2 7 2 7 7 7  No. of cores without tumor 3 2 3 3 3 3 3  Percentage 2 1 2 2 2 2 2 H&E 3 Table 3 Agreement in the degree of differentiation between TMA cores and full sections   Full section G1 G2 G3 Total κ TMA G1 2 3 0 5   G2 2 21 2 25   G3 0 7 26 33   Total 4 31 28 63 0.65 G1 G2 G3 1 4 Fig. 1 a b a c  Table 4 Agreement in immunohistochemical scores between TMA cores and full slides stained for CK5/6 and CK14   Full sections <10% 10–80% ≥80% Total κ TMAs CK5/6  <10% 1 1 0 2   10–80% 0 4 0 4   ≥80% 0 0 58 58   Total 1 5 58 64 0.93 CK14  <10% 5 2 1 8   10–80% 0 11 2 13   ≥80% 0 4 34 38   Total 5 17 37 59 0.71 4 5 Table 5 Agreement in immunohistochemical scores between TMA cores and full slides stained for E-cadherin     Full sections E-cadherin <50% ≥50% Total κ TMA  <50% 22 17 39   ≥50% 1 24 25   Total 23 41 64 0.47 6 Table 6 Agreement in immunohistochemical scores between TMA cores and full slides stained for Ki-67 and p53   Full sections <10% 10–50% ≥50% Total κ TMAs Ki-67  <10% 2 1 0 3   10–50% 3 42 3 48   ≥50% 0 6 4 10   Total 5 49 7 61 0.42 p53  <10% 19 3 0 22   10–50% 0 1 2 3   ≥50% 0 3 35 38  Total 19 7 37 63 0.86 6 Discussion 3 5 18 19 25 41 36 8 33 7 8 27 31 35 42 2 34 κ 4 7 8 31 35 31 6 11 12 28 12 42 33 The agreement between TMA and full sections was substantial to almost perfect for CK5/6 and CK14. Because 91% of cases have shown a very strong expression of CK5/6 and only 1 of 64 cases showed negative staining, this molecular marker does not subdivide ESCCs and consequently will not be a prognostic marker for this malignancy. CK14 was more evenly distributed over the three scoring groups, but because one case was scored two classes lower on TMA when compared to the full section, kappa was lower when compared to CK5/6. 2 Fig. 2 a b a c  κ 22 κ 3 Fig. 3 a b a c  7 31 κ 7 Now that our esophageal cancer TMA has been validated, it will be used to correlate the expression of various molecular pathways with clinicopathologic data, aiming at detecting markers of prognostic significance and molecular targets for new therapies. Because the agreement between TMA slides and full sections depended on the molecular marker stained for, it should be considered to assess the expression pattern of a marker on a full section first, before staining a TMA slide. When a focal or heterogeneous expression pattern is noticed, it might be more valuable to assess marker expression by means of full sections instead of TMA. On the other hand, when a marker shows a homogeneously diffuse expression pattern, staining a TMA slide does allow for high-throughput screening of tumors. When a prognostic molecular marker has been identified by means of TMA technology, it is recommended to verify the results by full-section analysis. In conclusion, this study has demonstrated TMA technology to be a valid method for immunohistochemical analysis in ESCC with agreement levels for well-known molecular markers with different staining potential between TMA and full sections ranging from moderate to almost perfect.