InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma

Introduction We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. We also evaluated tissue-derived predictors of extracted nucleic acids quality and success in downstream testing. Methods Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACT TM assay, methylation-profiling (array), and miRNA expression (Nanostring nCounter). Results Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p=0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p<0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001). Conclusion Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma.

引言 我们正在进行一项多中心研究，以确定原发性黑色素瘤患者疾病特异性生存的预测分类器。在此，我们阐述了针对包括来自美国癌症联合委员会（AJTCC）TNM分期为IIA - IIID患者的至少1.05mm原发性黑色素瘤在内的通常较小的色素性肿瘤样本进行优化研究的独特方面、挑战以及最佳实践。这项正在进行的研究将针对国际InterMEL联盟内的1000例黑色素瘤。我们还评估了组织来源的核酸提取质量预测因子以及下游检测的成功率。 方法 按照预先制定的方案，参与中心将福尔马林固定石蜡包埋（FFPE）组织切片送至纪念斯隆 - 凯特琳癌症中心，进行集中处理、皮肤病理检查以及在组织学指导下同时提取RNA和DNA。样本被分配用于通过使用MSK - IMPACT™检测的新一代测序（NGS）评估体细胞突变、甲基化分析（芯片）以及微小RNA（miRNA）表达（Nanostring nCounter）。 结果 在685例符合条件的黑色素瘤中，有683例（99%）获得了足够用于筛选miRNA表达的材料，467例（68%）用于甲基化分析，560例（82%）用于体细胞突变分析。在685例中的446例（65%）中，RNA/DNA的等份样本足以在所有三个平台上进行检测。在本次分析时已评估的样本中，平均NGS覆盖度为249x，59例（18.6%）样本覆盖度低于100x，41/414例（10%）由于低强度探针或不充分的Meta - Mixed四分位间距（BMIQ）和单样本（ss） - Noob标准化而未通过甲基化质量控制。683例RNA中的6例（1%）由于高于最低阈值的探针比例低而未通过Nanostring质量控制。FFPE组织块的年龄（p < 0.001）以及从切片到同时提取所经过的时间（p = 0.002）与甲基化筛选失败相关。黑色素降低了扩增200bp及以上片段的能力（无色素/轻度色素沉着与重度色素沉着相比，p < 0.003）。相反，重度色素沉着的肿瘤产生更多的RNA（p < 0.001）以及大于200个核苷酸的RNA（p < 0.001）。 结论 我们对许多存档组织的经验表明，通过对组织处理和质量控制的精心管理，在涉及少量FFPE肿瘤（如早期黑色素瘤研究）的复杂多机构环境中进行多组学研究是可能的。