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. 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

The Palaeoproterozoic Hotazel BIF-Mn Formation as an archive of Earth's earliest oxygenation

The 2.4Ga Hotazel Formation is a cyclically interlayered sequence of banded iron formation (BIF) and manganese-rich sedimentary rock at the uppermost part of the Neoarchaean-Palaeoproterozoic Transvaal Supergroup in South Africa. It represents an unusual stratigraphic association in the context of the origin of BIF and the coevolution of oxygen and life on early Earth and hence bears special relevance to the environmental conditions and processes that characterized the period leading up to the Great Oxidation Event (GOE) at ca. 2.3Ga. The mineral assemblages that characterize the Hotazel rocks are dominated by carbonate, silicate and oxide minerals, which are traditionally interpreted as predominantly diagenetic in origin, particularly the carbonates. By contrast, primary mineral assemblages are inferred to have been dominated by ferric oxyhydroxides and tetravalent manganese oxides, which show no preservation in the rock record and consequently hinder reconstruction of environmental conditions during sedimentation. Here, we revisit the Hotazel succession with a focus on its bulk-rock and carbonate-specific mineralogical, geochemical and stable isotope (C, Fe) composition by applying for the first time a high-resolution stratigraphic approach to sampling and analysis. Our main aim is to constrain the precursor mineralogy to the Fe- and Mn-rich facies in the Hotazel strata in order to unravel the redox conditions behind the massive cyclic deposition of Fe and Mn at the onset of the GOE. Our carbonate-specific results question traditional diagenetic models for the development of the carbonate fraction of the rocks and instead place the origin of much of the present mineralogy on water-column processes in a stratified basin characterized by successive redox pathways with changing water depth. These pathways exploited a series of thermodynamically predictable electron acceptors for organic carbon recycling, which included – probably for the first time in Earth history – aqueous Mn(III) and O2 as electron acceptors for the oxidation of both Fe(II) and organic carbon. The emergence of Mn(III) was also critical for the development of a Mn redox shuttle, which led to effective water-column stratification between aqueous Mn and Fe in the depositional basin. We conclude that the first known record of Mn(II) to Mn(III) oxidation as recorded in the Hotazel Formation must be a fundamentally diagnostic step in the redox evolution of the oceans and atmosphere in the lead-up to the GOE

Carbon isotope stratigraphy of Precambrian iron formations and possible significance for the early biological pump

The origin of Precambrian iron-formations (IF) remains contentious, particularly with respect to the mineralogy of primary precipitates and the exact processes and conditions leading to their formation. Despite the uncertainties, prevailing hypotheses range from biological precipitation of ferrihydrite to abiotic water-column formation of greenalite. By contrast, iron carbonate minerals (siderite, ankerite) in IF have traditionally been attributed to diagenetic origins based on textural and isotopic relationships. Recent studies on IF from the Neoarchaean-Paleoproterozoic Transvaal Supergroup of South Africa have revealed evidence for apparently primary, low-δ13C, Fe/Mn-bearing Mg calcite as precursor to iron carbonate formation and as a potentially underestimated pathway of isotopically light carbon burial during IF deposition. Here, we present whole-rock δ13C data and carbonate-specific geochemical analyses for samples from five drill cores that capture the entire stratigraphic extent of the Kuruman and Griquatown IF of the Transvaal Supergroup. Our results demonstrate remarkable consistency in stratigraphic profiles among the locations for the trends and magnitudes of bulk δ13C values that are independent of paragenetic association, modal mineralogy, and chemical composition of the bulk carbonate fraction of each sample. We interpret these records as resulting from water-column abiotic carbonate formation that was accompanied by kinetic isotopic effects associated with fluctuating conditions (pH, alkalinity) controlling carbonate supersaturation in ambient seawater. Although our interpretation provides strong support for abiotic, anoxic models for IF genesis prior to the Great Oxidation Event (GOE), it does not entirely preclude additional biological mechanisms of primary ferric oxyhydroxide formation and its possible role in an early biological pump

Carbon isotope stratigraphy of Precambrian iron formations and possible significance for the early biological pump

The origin of Precambrian iron-formations (IF) remains contentious, particularly with respect to the mineralogy of primary precipitates and the exact processes and conditions leading to their formation. Despite the uncertainties, prevailing hypotheses range from biological precipitation of ferrihydrite to abiotic water-column formation of greenalite. By contrast, iron carbonate minerals (siderite, ankerite) in IF have traditionally been attributed to diagenetic origins based on textural and isotopic relationships. Recent studies on IF from the Neoarchaean-Paleoproterozoic Transvaal Supergroup of South Africa have revealed evidence for apparently primary, low-δ13C, Fe/Mn-bearing Mg calcite as precursor to iron carbonate formation and as a potentially underestimated pathway of isotopically light carbon burial during IF deposition. Here, we present whole-rock δ13C data and carbonate-specific geochemical analyses for samples from five drill cores that capture the entire stratigraphic extent of the Kuruman and Griquatown IF of the Transvaal Supergroup. Our results demonstrate remarkable consistency in stratigraphic profiles among the locations for the trends and magnitudes of bulk δ13C values that are independent of paragenetic association, modal mineralogy, and chemical composition of the bulk carbonate fraction of each sample. We interpret these records as resulting from water-column abiotic carbonate formation that was accompanied by kinetic isotopic effects associated with fluctuating conditions (pH, alkalinity) controlling carbonate supersaturation in ambient seawater. Although our interpretation provides strong support for abiotic, anoxic models for IF genesis prior to the Great Oxidation Event (GOE), it does not entirely preclude additional biological mechanisms of primary ferric oxyhydroxide formation and its possible role in an early biological pump