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).