Richman Susan D, Hemmings Gemma, Roberts Helen, Gallop Niall, Dodds Rachel, Wilkinson Lyndsay, Davis Jonathan, White Rhian, Yates Emma, Jasani Bharat, Brown Louise, Maughan Tim S, Butler Rachel, Quirke Philip, Adams Richard
Leeds Institute on Medical Research, University of Leeds, Leeds, UK
Leeds Institute on Medical Research, University of Leeds, Leeds, UK.
J Clin Pathol. 2023 Aug;76(8):548-554. doi: 10.1136/jclinpath-2022-208233. Epub 2022 Mar 7.
FOCUS4 was a phase II/III umbrella trial, recruiting patients with advanced or metastatic colorectal cancer, between 2014 and 2020. Molecular profiling of patients' formalin-fixed, paraffin-embedded tumour blocks was undertaken at two centralised biomarker laboratories (Leeds and Cardiff), and the results fed directly to the Medical Research Council Clinical Trials Unit, and used for subsequent randomisation. Here the laboratories discuss their experiences.
Following successful tumour content assessment, blocks were sectioned for DNA extraction and immunohistochemistry (IHC). Pyrosequencing was initially used to determine tumour mutation status (KRAS, NRAS, BRAF and PIK3CA), then from 2018 onwards, next-generation sequencing was employed to allow the inclusion of TP53. Protein expression of MLH1, MSH2, MSH6, PMS2 and pTEN was determined by IHC. An interlaboratory comparison programme was initiated, allowing sample exchanges, to ensure continued assay robustness.
1291 tumour samples were successfully analysed. Assay failure rates were very low; 1.9%-3.3% for DNA sequencing and 0.9%-1.3% for IHC. Concordance rates of 98% were seen for the interlaboratory comparisons, where a result was obtained by both laboratories.
Practical and logistical problems were identified, including poor sample quality and difficulties with sample anonymisation. The often last-minute receipt of a sample for testing and a lack of integration with National Health Service mutation analysis services were challenging. The laboratories benefitted from both pretrial validations and interlaboratory comparisons, resulting in robust assay development and provided confidence during the implementation of new sequencing technologies. We conclude that our centralised approach to biomarker testing in FOCUS4 was effective and successful.
FOCUS4是一项II/III期伞式试验，在2014年至2020年期间招募晚期或转移性结直肠癌患者。在两个集中的生物标志物实验室（利兹和加的夫）对患者的福尔马林固定、石蜡包埋肿瘤块进行分子分析，结果直接反馈给医学研究理事会临床试验单位，并用于后续随机分组。在此，各实验室讨论了他们的经验。
在成功评估肿瘤含量后，将组织块切片用于DNA提取和免疫组织化学（IHC）。最初使用焦磷酸测序来确定肿瘤突变状态（KRAS、NRAS、BRAF和PIK3CA），然后从2018年起，采用下一代测序以纳入TP53。通过IHC确定MLH1、MSH2、MSH6、PMS2和pTEN的蛋白表达。启动了一项实验室间比较计划，允许样本交换，以确保检测的持续稳健性。
成功分析了1291个肿瘤样本。检测失败率非常低；DNA测序为1.9%-3.3%，IHC为0.9%-1.3%。在两个实验室都获得结果的实验室间比较中，一致性率为98%。
发现了实际和后勤方面的问题，包括样本质量差和样本匿名化困难。经常在最后一刻收到检测样本以及与国民医疗服务体系突变分析服务缺乏整合具有挑战性。各实验室从审前验证和实验室间比较中受益，从而实现了稳健的检测方法开发，并在新测序技术实施过程中提供了信心。我们得出结论，我们在FOCUS4中采用的集中式生物标志物检测方法是有效且成功的。
