Rapid Autopsy for Melanoma Profiling

Recruitment to the CASCADE (CAncer tiSsue aCquisition After DEath rapid autopsy programme at the Peter MacCallum Cancer Centre followed approved protocols (PMCC Human Research Ethics Committee approval number 11/102). Informed written consent was obtained from all patients and families. Rapid autopsies were performed as previously described^{22 (link)}. See Supplementary Methods for further details.
Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned 20 times and the 1st, 10th and 20th sections underwent haematoxylin & eosin (H&E), Melan-A and S100 staining, respectively, for identification by an expert histopathologist of intra-tumoural regions of viable melanoma. Only samples that contained >70% tumour content were used for DNA extraction and sequencing. After macro-dissection and DNA extraction (QIAamp DNA FFPE Tissue Kit, Qiagen), each sample was assessed for DNA integrity^{64 (link)}. In cases where adjacent fresh frozen samples were available for DNA extraction (DNeasy blood and tissue kit, Qiagen), tumour content was verified using digital PCR for BRAF mutations^{65 (link)}. Genomic DNA from buffy coats (used as matching germline DNA) was extracted using the DNeasy blood and tissue kit (Qiagen). Plasma DNA was extracted from 1–2 ml of plasma (QIAamp Circulating Nucleic Acid Kit, Qiagen).

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Vergara I.A., Mintoff C.P., Sandhu S., McIntosh L., Young R.J., Wong S.Q., Colebatch A., Cameron D.L., Kwon J.L., Wolfe R., Peng A., Ellul J., Dou X., Fedele C., Boyle S., Arnau G.M., Raleigh J., Hatzimihalis A., Szeto P., Mooi J., Widmer D.S., Cheng P.F., Amann V., Dummer R., Hayward N., Wilmott J., Scolyer R.A., Cho R.J., Bowtell D., Thorne H., Alsop K., Cordner S., Woodford N., Leditschke J., O’Brien P., Dawson S.J., McArthur G.A., Mann G.J., Levesque M.P., Papenfuss A.T, & Shackleton M. (2021). Evolution of late-stage metastatic melanoma is dominated by aneuploidy and whole genome doubling. Nature Communications, 12, 1434.

Publication 2021

QIAamp DNA FFPE Tissue Kit DNeasy blood and tissue kit QIAamp Circulating Nucleic Acid Kit

Autopsies Blood Braf Cancer Circulating nucleic acid Dissection Eosin Formalin Frozen Genomic Germline Haematoxylin Human Melan a Melanoma Paraffin Patients Plasma Research ethics committee S100 Tissue Tissue death Tumour

Corresponding Organization :

Other organizations : Walter and Eliza Hall Institute of Medical Research, Peter MacCallum Cancer Centre, Monash University, University Hospital of Zurich, University of Zurich, Melanoma Institute Australia, University of California, San Francisco, Victorian Institute of Forensic Medicine, University of Melbourne

Top 5 similar protocols

Variable analysis

independent variables

Recruitment to the CASCADE (CAncer tiSsue aCquisition After DEath rapid autopsy programme at the Peter MacCallum Cancer Centre

dependent variables

Identification of intra-tumoural regions of viable melanoma by an expert histopathologist
DNA integrity assessment
Tumor content verification using digital PCR for BRAF mutations

control variables

Sections used for H&E, Melan-A and S100 staining
Samples with >70% tumor content used for DNA extraction and sequencing
Matching germline DNA extracted from buffy coats
Plasma DNA extracted from 1-2 ml of plasma

controls

Positive control: Adjacent fresh frozen samples used for DNA extraction and tumor content verification
Negative control: Not explicitly mentioned

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