Supplement:
METHODS
Patient enrollment
Sequencing of clinical samples was performed under our Institutional Review Board (IRB)–approved studies at the University of Michigan (Michigan Oncology Sequencing Protocol, MI-ONCOSEQ, IRB # HUM00046018, HUM00067928, HUM00056496).
Integrative Clinical Sequencing
Two archival formalin-fixed paraffin-embedded (FFPE) samples and a frozen bone marrow sample were obtained for sequencing. A section of FFPE blocks were cut for evaluation, and remaining portions of each specimen were retained for nucleic acid extraction. Hematoxylin and eosin (H&E)-stained sections were reviewed by a board certified pathologist to identify areas with highest tumor content and an area of normal tissue. Tumor genomic DNA and total RNA from the frozen bone marrow were extracted using the AllPrep DNA/RNA/miRNA kit (QIAGEN). DNA from FFPE samples was extracted using the DNeasy Blood and Tissue Kit (QIAGEN), and total RNA was extracted using the miRNeasy FFPE kit (QIAGEN). RNA integrity was measured on an Agilent 2100 Bioanalyzer (Agilent Technologies).
Integrative clinical sequencing was performed in a Clinical Laboratory Improvement Amendments (CLIA) compliant sequencing lab as described before (Refs 4-5). Paired-end target-captured exome libraries from tumor and normal samples, and tumor transcriptome libraries were sequenced using the Illumina HiSeq2500. Aligned exome and transcriptome sequences were analyzed to detect somatic mutations, copy-number alterations, gene fusions, and gene expression as described before (Ref 4).