InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma
Authors: Orlow Irene , Sadeghi Keimya D. , Edmiston Sharon N. , Kenney Jessica M. , Lezcano Cecilia , Wilmott James S. , Cust Anne E. , Scolyer Richard A. , Mann Graham J. , Lee Tim K. , Burke Hazel , Jakrot Valerie , Shang Ping , Ferguson Peter M. , Boyce Tawny W. , Ko Jennifer S. , Ngo Peter , Funchain Pauline , Rees Judy R. , O’Connell Kelli , Hao Honglin , Parrish Eloise , Conway Kathleen , Googe Paul B. , Ollila David W. , Moschos Stergios J. , Hernando Eva , Hanniford Douglas , Argibay Diana , Amos Christopher I. , Lee Jeffrey E. , Osman Iman , Luo Li , Kuan Pei-Fen , Aurora Arshi , Gould Rothberg Bonnie E. , Bosenberg Marcus W. , Gerstenblith Meg R. , Thompson Cheryl , Bogner Paul N. , Gorlov Ivan P. , Holmen Sheri L. , Brunsgaard Elise K. , Saenger Yvonne M. , Shen Ronglai , Seshan Venkatraman , Nagore Eduardo , Ernstoff Marc S. , Busam Klaus J. , Begg Colin B. , Thomas Nancy E. , Berwick Marianne ,   on behalf of the InterMEL Consortium
Published: April 2023

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. We also evaluated tissue-derived predictors of extracted nucleic acids’ quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium.

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-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay).

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. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.