Paolo Bironzo, Laura Melocchi, Valentina Monica, Dario Trebeschi, Fausto Barbieri, Evaristo Maiello, Maria Rita Migliorino, Alessandra Lombardi, Marcello Tiseo, Luisella Righi, Paolo Graziano, Giulio Rossi, Silvia Novello
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TS expression by real time (RT)-PCR was compared with <i>ALK</i> FISH-negative cases.</p><p><strong>Materials and methods: </strong>37 ALK FISH-positive NSCLC cases diagnosed between 2010 and 2015 in 7 Italian centres were investigated with ICH using three different anti-ALK antibodies (ALK1, 5A4 and D5F3). Staining for ALK1 and 5A4 was graded as 0+,1+,2+, and 3+, while the scoring for D5F3 was recorded as negative or positive. Proportion agreement analysis was done using Cohen's unweighted kappa (k). TS and β-actin expression levels were analysed by quantitative RT-PCR. Comparison between TS expression in ALK FISH-positive specimens and a control cohort of ALK FISH-negative ones was performed with the Mann-Whitney and Kruskal-Wallis tests.</p><p><strong>Results: </strong>Considering 2+ and 3+ as positive, the proportion of IHC agreement was 0.1691 (95% CI 0-0.4595) for ALK1/5A4, 0.1691 (95% CI 0-0.4595) for ALK1/D5F3, and 1 for D5F3/5A4. Considering 3+ as positive, it was 0.1543 (95% CI 0-0.4665) for ALK1/ 5A4, 0.0212 (95% CI 0-0.1736) for ALK1/D5F3, and 0.2269 (95% CI 0-0.5462) for 5A4/D5F3. Median TS expression was 6.07 (1.28-14.94) and ALK-positive cases had a significant lower TS expression than ALK-negative tumours <i>(p = 0.002).</i></p><p><strong>Conclusions: </strong>IHC proved to be a reliable tool for the diagnosis of ALK-rearranged NSCLC. D5F3 and 5A4 clones have the highest percentage of agreement. TS levels are significantly lower in FISH-positive patients.</p>","PeriodicalId":45893,"journal":{"name":"PATHOLOGICA","volume":"114 4","pages":"278-287"},"PeriodicalIF":4.4000,"publicationDate":"2022-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/39/8c/pathol-2022-04-278.PMC9624140.pdf","citationCount":"0","resultStr":"{\"title\":\"Immunohistochemistry with 3 different clones in anaplastic lymphoma kinase fluorescence in situ hybridization positive non-small-cell lung cancer with thymidylate synthase expression analysis: a multicentre, retrospective, Italian study.\",\"authors\":\"Paolo Bironzo, Laura Melocchi, Valentina Monica, Dario Trebeschi, Fausto Barbieri, Evaristo Maiello, Maria Rita Migliorino, Alessandra Lombardi, Marcello Tiseo, Luisella Righi, Paolo Graziano, Giulio Rossi, Silvia Novello\",\"doi\":\"10.32074/1591-951X-756\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Introduction: </strong><i>ALK</i> rearrangement is the only druggable oncogenic driver detectable by immunohistochemistry (IHC) not requiring further confirmation of positivity in accessing first-line specific inhibitors. 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Comparison between TS expression in ALK FISH-positive specimens and a control cohort of ALK FISH-negative ones was performed with the Mann-Whitney and Kruskal-Wallis tests.</p><p><strong>Results: </strong>Considering 2+ and 3+ as positive, the proportion of IHC agreement was 0.1691 (95% CI 0-0.4595) for ALK1/5A4, 0.1691 (95% CI 0-0.4595) for ALK1/D5F3, and 1 for D5F3/5A4. Considering 3+ as positive, it was 0.1543 (95% CI 0-0.4665) for ALK1/ 5A4, 0.0212 (95% CI 0-0.1736) for ALK1/D5F3, and 0.2269 (95% CI 0-0.5462) for 5A4/D5F3. Median TS expression was 6.07 (1.28-14.94) and ALK-positive cases had a significant lower TS expression than ALK-negative tumours <i>(p = 0.002).</i></p><p><strong>Conclusions: </strong>IHC proved to be a reliable tool for the diagnosis of ALK-rearranged NSCLC. D5F3 and 5A4 clones have the highest percentage of agreement. 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引用次数: 0
摘要
ALK重排是免疫组化(IHC)检测到的唯一可用药的致癌驱动因素，在获得一线特异性抑制剂时无需进一步确认阳性。alk阳性患者从培美曲塞为基础的化疗中获益，可能是由于胸腺苷酸合成酶(TS)水平较低。本研究在37例fish阳性NSCLC中评估了三种不同的ALK IHC克隆的一致性。实时(RT)-PCR法比较ALK - fish阴性患者的TS表达情况。材料和方法:2010年至2015年间，意大利7个中心诊断的37例ALK fish阳性非小细胞肺癌患者使用三种不同的抗ALK抗体(ALK1, 5A4和D5F3)进行ICH调查。ALK1和5A4染色分为0+、1+、2+和3+，D5F3评分为阴性或阳性。采用Cohen's未加权kappa (k)进行比例一致性分析。采用定量RT-PCR分析TS和β-actin表达水平。采用Mann-Whitney和Kruskal-Wallis试验比较ALK fish阳性标本和ALK fish阴性对照标本中TS的表达。结果:考虑2+和3+为阳性，ALK1/ 5a4的IHC一致性比例为0.1691 (95% CI 0-0.4595)， ALK1/D5F3为0.1691 (95% CI 0-0.4595)， D5F3/ 5a4为1。考虑到3+为阳性，ALK1/ 5A4为0.1543 (95% CI 0-0.4665)， ALK1/D5F3为0.0212 (95% CI 0-0.1736)， 5A4/D5F3为0.2269 (95% CI 0-0.5462)。TS的中位表达量为6.07 (1.28-14.94)，alk阳性患者的TS表达量显著低于alk阴性患者(p = 0.002)。结论:免疫组化是诊断alk重排非小细胞肺癌的可靠工具。D5F3和5A4克隆的一致性百分比最高。fish阳性患者的TS水平显著降低。
Immunohistochemistry with 3 different clones in anaplastic lymphoma kinase fluorescence in situ hybridization positive non-small-cell lung cancer with thymidylate synthase expression analysis: a multicentre, retrospective, Italian study.
Introduction: ALK rearrangement is the only druggable oncogenic driver detectable by immunohistochemistry (IHC) not requiring further confirmation of positivity in accessing first-line specific inhibitors. ALK-positive patients experience clinical benefit from pemetrexed-based chemotherapy possibly due to lower thymidylate synthase (TS) levels. This study assesses agreement with three different ALK IHC clones in 37 FISH-positive NSCLC. TS expression by real time (RT)-PCR was compared with ALK FISH-negative cases.
Materials and methods: 37 ALK FISH-positive NSCLC cases diagnosed between 2010 and 2015 in 7 Italian centres were investigated with ICH using three different anti-ALK antibodies (ALK1, 5A4 and D5F3). Staining for ALK1 and 5A4 was graded as 0+,1+,2+, and 3+, while the scoring for D5F3 was recorded as negative or positive. Proportion agreement analysis was done using Cohen's unweighted kappa (k). TS and β-actin expression levels were analysed by quantitative RT-PCR. Comparison between TS expression in ALK FISH-positive specimens and a control cohort of ALK FISH-negative ones was performed with the Mann-Whitney and Kruskal-Wallis tests.
Results: Considering 2+ and 3+ as positive, the proportion of IHC agreement was 0.1691 (95% CI 0-0.4595) for ALK1/5A4, 0.1691 (95% CI 0-0.4595) for ALK1/D5F3, and 1 for D5F3/5A4. Considering 3+ as positive, it was 0.1543 (95% CI 0-0.4665) for ALK1/ 5A4, 0.0212 (95% CI 0-0.1736) for ALK1/D5F3, and 0.2269 (95% CI 0-0.5462) for 5A4/D5F3. Median TS expression was 6.07 (1.28-14.94) and ALK-positive cases had a significant lower TS expression than ALK-negative tumours (p = 0.002).
Conclusions: IHC proved to be a reliable tool for the diagnosis of ALK-rearranged NSCLC. D5F3 and 5A4 clones have the highest percentage of agreement. TS levels are significantly lower in FISH-positive patients.
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