Supplementary figure legends
Supplementary Figure 1 – DNA sequence of DICER1 exon 23 from blood and tumor
specimens using discriminating primers across germline mutant sequence
(c.4407_4410delTTCT). Tumor DNA shows c.5125G>A (p.D1709N) in wild-type allele
(germline mutant-negative allele).
Supplementary Figure 2 – Expression levels of miRNAs with at least three-fold
difference in median expression between PPB and normal lung tissues from DICER1
germline carriers. Hierarchical clustering suggests a depletion of 5’-derived miRNAs
relative to 3’-derived miRNAs in PPB tumors with DICER somatic missense mutations
in addition to germline loss-of-function variants.
Supplementary Figure 3 – qRT-PCR from DICER1 mutant tumors demonstrating
decreased expression of 5p-microRNA in PPB (type II and III) patient tissues. Five
members of let-7 family including miR-98, plus miR-34a and c shown.
Supplementary Figure 4 – Distribution of sequence coverage across each miRNA,
normalized for the length of each miRNA primary transcript with at least one sequencing
read aligned. Columns correspond to different subsets of expressed miRNAs. All
miRNAs: All miRNAs with a 5p, 3p, or 5p and 3p arms annotated in miRbase build 19.
Two arms: Only miRNAs with 5p and 3p arms annotated. One arm: miRNAs with a
single arm annotated in the first 50% (5p) or latter 50% (3p) of the primary transcript.
Each row corresponds to a different sample subject to miRNA sequencing. Normal:
Reads from normal fibroblasts mapped predominantly to mature 5p and 3p miRNA
transcripts resulting in a characteristic “valley” of lower coverage of cleaved and
degraded hairpin sequence. PPB_14 tumor: Coverage profiles from primary PPB tissue
have increased coverage of hairpin sequences. “One arm” 5p miRNAs have increased
coverage beyond the boundary of annotated 5p miRNA region whereas profiles from
one-arm 3p miRNAs are more similar to normal tissue. PPB_14 cell line: In the
presumably clonal tumor cell line, hairpin sequences are retained to a greater degree than
the heterogeneous tumor tissue specimen. This suggests the small hairpin “valley” seen in
PPB_14 tumor may be due to infiltrating normal cells. As with the tumor sample, the
retention appears to be specific to 5p-derived miRNAs as one-arm 3p miRNAs do not
have significant coverage beyond the annotated boundary of mature transcripts. miRNA
arm pos: Line segments depicting the location of each annotated mature transcript
mapped to the percent location along each miRNA primary transcript. Transcripts with
both 5p and 3p arms annotated are marked in black and red. Transcripts with only a
single annotated mature transcript are marked in blue. Y-axis is a count miRNAs in each
miRNA subset.
Supplementary Figure 5 – Photomicrographs of PPB tumors stained with anti-p53
antibodies shows diffuse positivity, subclonal positivity or negative staining. Results
compared with mutation analysis performed on DNA extracted from a 5mm core from
the same FFPE tumor block. Anti-p53 rabbit monoclonal with hematoxylin counterstain;
original magnification x 200 A, C and D; x 400 B. A) PPB_5 tumor shows strong
nuclear positivity in the majority of tumor cells. Exome sequence analysis identified an
exon 5 c.481G>A; p.A161T missense mutation in TP53 predicted to be deleterious by
SIFT; B) PPB_16 tumor is negative for p53. Ion Torrent sequence analysis identified an
exon 5 c.513-514 insA frameshift mutation in TP53; C) PPB_11 tumor was also negative
for p53. This tumor had an exon 6 c. 642_643delTA frameshift mutation and deletion of
the wild-type allele identified by exome sequencing; D) PPB_17 tumor shows diffuse
anaplasia with focal areas of cells with nuclear p53 staining approximating 20% of the
tumor. Ion Torrent sequencing showed an exon 5 c.481G>A; p.A161T missense mutation
supported by 7% of reads. We interpret the immunohistochemistry result as a mixed
population of cells with 20% mutant TP53/deletion TP53 and 80% cells with biallelic
TP53 deletion with the mutant TP53 population not represented in the core taken for
genetic analysis (region of tumor selected by H&E only). Note the cytologic features of
the p53 positive nuclei and p53 negative nuclei are identical showing significant
anaplasia.
Supplementary Figure 6 – Fraction of read counts with start and end points confined to
functional units of 1,595 miRNA primary transcripts. 5p and 3p regions as annotated by
miRbase build 19. Hairpin loop regions were defined as the genome segment between 5p
and 3p regions. 5p+loop+3p denotes reads that include 5p, loop, and 3p sequence. The
“Unannotated” category denotes miRNAs without an explicit 5p or 3p designation. On
each page, the three rows correspond to the three samples presented in Figure 4: a normal
fibroblast cell line, a PPB primary tumor, and a PPB cell line derived from the PPB
primary tumor tissue. miRNAs are ordered by the number of reads in the normal sample.
Supplementary table descriptions
Supplementary Table 1 – Matrix of clinical features, copy number summary, and
mutation calls by case.
Supplementary Table 2 – List of annotated somatic mutation calls.
Supplementary Table 3 – Somatic mutation significance table (MutSig v1.5).
Supplementary Table 4 – Summary of somatic DICER1 mutations in discovery and
extension cohorts
Supplementary Table 5 – Summary of samples analyzed by quantative RT-PCR to
support Supplemental Figure 3
Supplementary Table 6 – Summary of somatic TP53 mutations and
immunohistochemistry patterns in 27 tumors
Supplementary Table 7 – Broad somatic copy number variant significance table
(GISTIC2).
Supplementary Table 8 – Focal somatic copy number variant significance table
(GISTIC2).
Supplementary Table 9 - Counts of reads with start and end points confined to functional
units of 1,595 miRNA primary transcripts across a normal fibroblast cell line, a PPB
primary tumor, and a PPB cell line derived from the PPB primary tumor tissue. These are
the raw data that underlie panels B-D of Figure 4 and Supplementary Figure 6.