InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma
Orlow, I.; Sadeghi, K. D.; Edmiston, S. N.; Kenney, J. M.; Lezcano, C.; Wilmott, J. S.; Cust, A. E.; Scolyer, R. A.; Mann, G. J.; Lee, T. K.; Burke, H.; Jakrot, V.; Shang, P.; Ferguson, P. M.; Boyce, T. W.; Funchain, P.; Ko, J. S.; Ngo, P.; Rees, J. R.; OConnell, K.; Hao, H.; Parrish, E.; Conway, K.; Googe, P. B.; Ollila, D. W.; Moschos, S. J.; Hernando, E.; Hanniford, D.; Argibay, D.; Amos, C. I.; Lee, J. E.; Osman, I.; Luo, L.; Kuan, P.-F.; Aurora, A.; Gould Rothberg, B. E.; Bosenberg, M. W.; Gerstenblith, M. R.; Thompson, C.; Bogner, P. N.; Gorlov, I. P.; Holmen, S. L.; Brunsgaard, E. K.; S
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IntroductionWe 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. MethodsFollowing 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 assay, methylation-profiling (array), and miRNA expression (Nanostring nCounter). ResultsSufficient 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). ConclusionOur 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.
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