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-IMPACT TM 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.
SHOW FULL ABSTRACT Irene Orlow, Keimya D. Sadeghi, Sharon N. Edmiston, Jessica M. Kenney, Cecilia Lezcano, James S. Wilmott, Anne E. Cust, Richard A. Scolyer, Graham J. Mann, Tim K. Lee, Hazel Burke, Valerie Jakrot, Ping Shang, Peter M. Ferguson, Tawny W. Boyce, Jennifer S. Ko, Peter Ngo, Pauline Funchain, Judy R. Rees, Kelli O’Connell, Honglin Hao, Eloise Parrish, Kathleen Conway, Paul B. Googe, David W. Ollila, Stergios J. Moschos, Eva Hernando, Douglas Hanniford, Diana Argibay, Christopher I. Amos, Jeffrey E. Lee, Iman Osman, Li Luo, Pei-Fen Kuan, Arshi Aurora, Bonnie E. Gould Rothberg, Marcus W. Bosenberg, Meg R. Gerstenblith, Cheryl Thompson, Paul N. Bogner, Ivan P. Gorlov, Sheri L. Holmen, Elise K. Brunsgaard, Yvonne M. Saenger, Ronglai Shen, Venkatraman Seshan, Eduardo Nagore, Marc S. Ernstoff, Klaus J. Busam, Colin B. Begg, Nancy E. Thomas, Marianne Berwick
1
Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America 2
Department of Dermatology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America 3
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America 4
Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America 5
Melanoma Institute Australia, The University of Sydney, Sydney, New South Wales, Australia 6
Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia 7
Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital and NSW Health Pathology, Sydney, New South Wales, Australia 8
Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia 9
The Daffodil Centre, University of Sydney, a joint venture with Cancer Council New South Wales, Australia 10
Sydney School of Public Health, The University of Sydney, Sydney, Australia 11
Centre for Cancer Research, Westmead Institute for Medical Research, The University of Sydney, Westmead, New South Wales, Australia 12
John Curtin School of Medical Research, Australian National University, Canberra, Australia 13
British Columbia Cancer Research Center, Vancouver, British Columbia, Canada 14
Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital and New South Wales Health Pathology, Sydney, New South Wales, Australia 15
Department of Internal Medicine, University of New Mexico Comprehensive Cancer Center, Albuquerque, New Mexico, United States of America 16
Department of Pathology, Cleveland Clinic, Cleveland, Ohio, United States of America 17
Department of Hospital Medicine, Cleveland Clinic, Cleveland, Ohio, United States of America 18
Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio, United States of America 19
Department of Epidemiology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America 20
Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America 21
Department of Surgery, Division of Surgical Oncology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America 22
Department of Medicine, Division of Medical Oncology, The University of North Carolina Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina, United States of America 23
Department of Pathology, New York University Grossman School of Medicine, New York, New York, United States of America 24
Department of Medicine, Baylor College of Medicine, Houston, Texas, United States of America 25
Department of Surgical Oncology, University of Texas, MD Anderson Cancer Center, Houston, Texas, United States of America 26
Department of Urology, New York University Grossman School of Medicine, New York, NY, United States of America 27
Department of Medicine, New York University Grossman School of Medicine, New York, NY, United States of America 28
Department of Dermatology, New York University Grossman School of Medicine, New York, NY, United States of America 29
Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, New York, United States of America 30
Smilow Cancer Hospital, Yale-New Haven Health System, New Haven, Connecticut, United States of America 31
Department of Internal Medicine, Section of Medical Oncology, Yale School of Medicine, New Haven, Connecticut, United States of America 32
Department of Dermatology, Yale School of Medicine, New Haven, Connecticut, United States of America 33
Department of Pathology, Yale School of Medicine, New Haven, Connecticut, United States of America 34
Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut, United States of America 35
Department of Dermatology, University Hospitals Cleveland Medical Center/Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America 36
Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America 37
Department of Nutrition, Case Western Reserve University, Cleveland, Ohio, United States of America 38
Department of Public Health Sciences, Penn State University College of Medicine, Hershey, Pennsylvania, United States of America 39
Department of Dermatology, Roswell Park Comprehensive Cancer Center, Buffalo, New York, United States of America 40
Department of Pathology, Roswell Park Comprehensive Cancer Center, Buffalo, New York, United States of America 41
Department of Oncological Sciences, University of Utah Health Sciences Center, Salt Lake City, Utah, United States of America 42
Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, Utah, United States of America 43
Department of Surgery, University of Utah Health Sciences Center, Salt Lake City, Utah, United States of America 44
Department of Medicine, Division of Hematology/Oncology, Columbia University Irving Medical Center, New York, New York, United States of America 45
Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York, United States of America 46
Department of Dermatology, Instituto Valenciano de Oncologia, Valencia, Spain 47
Division of Cancer Treatment and Diagnosis, National Cancer Institute, ImmunoOncology Branch, Developmental Therapeutics Program, Rockville, Maryland, United States of America Fondazione IRCCS Istituto Nazionale dei Tumori, ITALY EDITOR / REVIEWER
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