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™ 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. ### Competing Interest Statement I have read the journal's policy and the authors of this manuscript have the following competing interests: R.A.S. has received fees for professional services from F. Hoffmann-La Roche Ltd, Evaxion, Provectus Biopharmaceuticals Australia, Qbiotics, Novartis, Merck Sharp & Dohme, NeraCare, AMGEN Inc., Bristol-Myers Squibb, Myriad Genetics, GlaxoSmithKline. ### Funding Statement This work was supported by the National Cancer Institute (NCI) at the National Institutes of Health (NIH), grant P01CA206980, the Cancer Center Support grants P30CA118100 (to University of New Mexico Comprehensive Cancer Center), P30CA008748 (to Memorial Sloan Kettering Cancer Center), P30 CA016087 (to the Laura and Isaac Perlmutter Cancer Center at NYULH), P30CA042014 (to Huntsman Cancer Institute Comprehensive Cancer Center), awards R01CA121118 and R21CA245577 (to S.L.H.), the Melanoma SPORE P50 CA225450 (to E.H. and I.O.), K08CA151645 (to B.E.G.R) the Australian National Health and Medical Research Council Investigator Grants 1174325 (to J.S.W.), 2008454 (to A.E.C.) the Australian National Health and Medical Research Council Practitioner Fellowship APP1141295 (to R.A.S.). Additional funding support was received from: The University of Sydney Faculty of Medicine and Health (to J.S.W. and R.A.S), the Huntsman Foundation (to S.L.H), The Char and Chuck Fowler Family Foundation (to M.R.G. and C.L.T), the Cleveland Foundation (to M.R.G.), the Roswell Park Alliance Foundation (to P.N.B.), The University of Texas MD Anderson Cancer Center Miriam and Jim Mulva Research Fund, The McCarthy Skin Cancer Research Fund, The Marit Peterson Fund for Melanoma Research, and The Irving and Nadine Mansfield and Robert David Levitt Cancer Research Chair (to J.E.L.), The Robert E. Leet and Clara Guthrie Patterson Trust (to B.E.G.R.). Funds from the National Institute of General Medical Sciences under award 1T32GM135128 (to S.N.E.), Cycle for Survival, the Marie-Josée and Henry R. Kravis Center for Molecular Oncology (The MSK Integrated Genomics Operation Core). The Ainsworth Foundation, ClearBridge Foundation, The Cameron Family, as well as from colleagues at Melanoma Institute Australia and Royal Prince Alfred Hospital is also gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study protocol was approved by the Institutional Review Boards (IRBs) at each participating institution (listed below), material and data user agreements are in place, and research has been conducted according to the principles expressed in the Declaration of Helsinki. Contributing Institutions: Cleveland Clinic (Cleveland, OH), Case Western Reserve University (Cleveland, OH), Dartmouth Cancer Center (Lebanon, NH), the University of Texas MD Anderson Cancer Center (Houston, TX), Melanoma Institute Australia (Sydney, NSW, Australia), Memorial Sloan Kettering Cancer Center (New York, NY), New York University Langone Medical Center (New York, NY), The University of North Carolina (Chapel Hill, NC, hereafter denoted UNC), Roswell Park Comprehensive Cancer Center (Buffalo, NY),Yale School of Medicine (New Haven, CT). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The data underlying this report are subject to an embargo until December 1st, 2023. Once the embargo expires, the data will be available on the National Cancer Institute dbGaP website upon request.