Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands.
Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Centers, VU University Amsterdam, Amsterdam, The Netherlands.
Lung Cancer. 2023 Apr;178:108-115. doi: 10.1016/j.lungcan.2023.02.007. Epub 2023 Feb 11.
The objective is to assess the impact of the quality of tissue fixation in surgical pathology on immunohistochemical (IHC) staining and DNA degradation.
Twenty-five non-small cell lung cancer (NSCLC) resection specimens were analyzed. After resection, all tumors were processed according to the protocols in our center. In haematoxylin and eosin (H&E) stained tissue slides, adequately- and inadequately fixed tumor areas were microscopically demarcated, based on basement membrane detachment. In 10 IHC stains ALK (clone 5A4), PD-L (clone 22C3), CAM5.2, CK7, c-Met, KER-MNF116, NapsinA, p40, ROS1, TTF1) the immunoreactivity in H-scores was determined in adequately- and inadequately fixed, and necrotic tumor areas. From the same areas DNA was isolated, and DNA fragmentation in base pairs (bp) was measured.
H-scores were significantly higher in H&E adequately fixed tumor areas in IHC stains KER-MNF116 (H-score 256 vs 15, p=0.001) and p40 (H-score 293 vs 248, p=0.028). All other stains showed a trend towards higher immunoreactivity in H&E adequately fixed areas. Independent of H&E adequatelty- or inadequately fixed areas, all IHC stains showed significant different IHC staining intensity within tumors, suggesting heterogeneous immunoreactivity (H-scores: PD-L1 123 vs 6, p = 0.001; CAM5.2 242 vs 101, p=<0.001; CK7 242 vs 128, p=<0.001; c-MET 99 vs 20, p=<0.001; KER-MNF116 281 vs 120, p=<0.001; Napsin A 268 vs 130, p = 0.005; p40 292 vs 166, p = 0.008; TTF1 199 vs 63, p=<0.001). DNA fragments rarely exceeded a length of 300 bp, independent of adequate fixation. However, DNA fragments of 300 and 400 bp had higher concentrations in tumors with short fixation delay (<6 h vs >16 h) and short fixation time (<24 h vs >24 h).
Impaired tissue fixation of resected lung tumors results in decreased IHC staining intensity in some parts of the tumor. This may impact the reliability of IHC analysis.
评估外科病理学中组织固定质量对免疫组织化学（IHC）染色和 DNA 降解的影响。
分析了 25 例非小细胞肺癌（NSCLC）切除标本。切除后，所有肿瘤均按照我们中心的方案进行处理。在苏木精和伊红（H&E）染色的组织切片中，根据基膜脱离，在显微镜下对充分固定和固定不足的肿瘤区域进行了微观标记。在 10 种 IHC 染色（ALK（克隆 5A4）、PD-L（克隆 22C3）、CAM5.2、CK7、c-Met、KER-MNF116、NapsinA、p40、ROS1、TTF1）中，确定了充分固定和固定不足以及坏死肿瘤区域的 H 评分中的免疫反应性。从同一区域分离 DNA，并测量碱基对（bp）的 DNA 片段化。
在 IHC 染色 KER-MNF116（H 评分 256 对 15，p=0.001）和 p40（H 评分 293 对 248，p=0.028）中，H&E 充分固定肿瘤区域的 H 评分明显更高。所有其他染色都显示出在 H&E 充分固定区域内更高的免疫反应性趋势。无论 H&E 是否充分固定，所有 IHC 染色在肿瘤内均显示出明显不同的 IHC 染色强度，表明存在异质性免疫反应（H 评分：PD-L1 123 对 6，p=0.001；CAM5.2 242 对 101，p<0.001；CK7 242 对 128，p<0.001；c-MET 99 对 20，p<0.001；KER-MNF116 281 对 120，p<0.001；Napsin A 268 对 130，p=0.005；p40 292 对 166，p=0.008；TTF1 199 对 63，p<0.001）。无论固定是否充分，DNA 片段很少超过 300bp 的长度。然而，在固定延迟时间较短（<6 小时对>16 小时）和固定时间较短（<24 小时对>24 小时）的肿瘤中，300bp 和 400bp 的 DNA 片段具有更高的浓度。
切除的肺肿瘤组织固定不良会导致肿瘤某些部位的 IHC 染色强度降低。这可能会影响 IHC 分析的可靠性。
