CDH1 truncating alterations were detected in all six plasmacytoid-variant bladder tumors analyzed by whole-exome sequencing.

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1 Supplementary Figure 1 CDH1 truncating alterations were detected in all six plasmacytoid-variant bladder tumors analyzed by whole-exome sequencing. Whole-exome sequencing of six plasmacytoid-variant bladder tumors (left) with copy number analysis of the CDH1 locus for two representative cases (right). All six tumors analyzed harbored truncating loss-of-function mutations in CDH1. Evidence for copy-neutral loss of the wild-type allele and loss of heterozygosity was observed in the two samples shown (asterisks above heat map). 1

2 Supplementary Figure 2 Pattern of CDH1 alterations and select co-alterations in plasmacytoid-variant bladder cancers, lobular breast carcinomas and diffuse gastric carcinomas. With the exception of CDH1 alterations, the pattern of genetic co-alterations in lobular breast and diffuse gastric carcinomas was distinct from that of plasmacytoid-variant bladder cancer. Somatic mutation data for the lobular breast carcinoma and diffuse gastric carcinomas was derived from The Cancer Genome Atlas. 2

3 Supplementary Figure 3 Phylogenetic tree depicting the divergent evolution of plasmacytoid-variant carcinoma and urothelial carcinoma, NOS, histologies within a tumor with distinct histologic regions. Exon capture and deep sequencing of two adjacent portions of a bladder tumor harboring distinct regions of plasmacytoid-variant and urothelial carcinoma, NOS, histologies. CDKN2A and PIK3C2G alterations were shared by plasmacytoid-variant histology and urothelial carcinoma, NOS, areas within the same tumor, suggesting that these are truncal alterations occurring within a common precursor cell. However, a CDH1 frameshift mutation was unique to plasmacytoid-variant histology, and the remaining genetic alteration profiles of the two histologic components were distinct. Magenta and green lines in the phylogenetic tree indicate plasmacytoid-variant and urothelial carcinoma, NOS, histologies, respectively. 3

4 Supplementary Figure 4. CDH1 promoter hypermethylation is a mechanism for loss of expression of E-cadherin in plasmacytoid-variant bladder cancer. Bisulfite sequencing of the CDH1 promoter CpG island showed hypermethylation in four of five CDH1 wild type plasmacytoid-variant bladder tumors. Representative urothelial carcinoma, NOS, specimens (U1, U2) that were subjected to bisulfite sequencing are shown to lack CDH1 promoter hypermethylation. A schematic of the CpG island is depicted with red circles representing methylated CpG sites. 4

5 Supplementary Figure 5 E-cadherin expression is absent in the invasive component of plasmacytoid-variant tumors. Low magnification H&E showing the in situ and invasive regions of plasmacytoid-variant tumors is displayed on the left. Highmagnification views with E-cadherin IHC of each of these regions are shown on the right. E-cadherin expression is retained within the in situ component and is absent from the invasive regions of this tumor. 5

6 Supplementary Table 1: CDH1 alterations identified in plasmacytoid variant bladder cancer ID Age/sex No of mutations CDH1 mutation E-cadherin IHC invasive region E-cadherin IHC in situ region Source of germline DNA 1 75/F 19 E58* 0 NA Benign pelvic lymph node 2 50/M 10 E806* 0 NA Benign pelvic lymph node 3 ǂ 72/F 8 Q129* 0 Membranous Benign pelvic lymph node 4 60/F 12 E648* 0 NA Benign pelvic lymph node 5 ǂ 57/F 8 Q677* 0 NA Benign pelvic lymph node 6 58/F 7 Q647* 0 NA Benign pelvic lymph node 7 72/F 23 E35* 0 NA Benign pelvic lymph node 8 40/F 17 W4* 0 NA Benign ovarian tissue 9 48/M 5 Q129* 0 Membranous Benign pelvic lymph node 10 ǂ 67/M 14 S70Pfs*13 0 NA Benign pelvic lymph node 11 ǁ 67/M 4 WT Membranous NA Benign pelvic lymph node 12 60/M 6 C163Afs*52 0 Membranous Benign pelvic lymph node 13 70/M 5 T366Rfs*5 0 NA Benign pelvic lymph node 14 68/F 9 E386K, F462Hfs*16 0 Membranous Benign cervical tissue 15 ǂ 69/M 7 F730Vfs*18 0 Membranous Benign skin from biopsy 16 67/M 13 X178_splice 0 NA Benign pelvic lymph node 17 54/F 4 X522_splice 0 NA Benign pelvic lymph node 18 ǂ 63/F 7 X522_splice 0 Membranous Benign small bowel segment 19 55/M 7 X441_splice Membranous Membranous Benign pelvic lymph node 20 56/M 23 V157F Weak cytoplasmic NA Benign pelvic lymph node 21 ǂ 73/M 6 S9*, D288N 0 Membranous Benign pelvic lymph node 22 83/M 5 WT, Hypermethylated 0 NA Benign prostatic tissue 23 71/M 2 WT 0 Membranous Benign pelvic lymph node 24 59/M 11 WT, Hypermethylated 0 Membranous Benign pelvic lymph node 25 76/M 30 WT, Hypermethylated 0 Membranous Benign pelvic lymph node 26 54/M 1 WT, Hypermethylated 0 NA Benign pelvic lymph node 27 Ɨ 60/M 18 E247* 0 Membranous Peripheral blood 28 Ɨ 66/F 8 R598* 0 NA Peripheral blood 29 Ɨ 84/M 9 Q23* 0 Membranous Peripheral blood 30 Ɨ 55/M 8 Q346*, Q351*, P373A 0 Membranous Peripheral blood 31 Ɨ 52/M 8 deletion: c.134_ del 0 Membranous Peripheral blood 32 Ɨ 36/M 118 E210Q 0 Membranous Peripheral blood Ɨ: Prospective cohort samples ǂ: Whole exome sequenced samples; WT: Wild type; M: Male; F: Female ǁ: Samples 10 and 11 are derived from the same tumor. 10 displays plasmacytoid features and 11 displays urothelial carcinoma, NOS features (see Supplemental Figure 3)

7 Supplementary Table 2: Primer sequences for bisulfite sequencing and CRISPR nickase Bisulfite sequencing primers for CDH1 5'-GGTAGGTGAATTTTTAGTTAATTAG-3' 5 -AATACCTACAACAACAACAACAAC-3' CDH1 exon 3 oligonucleotides used for LentiCRISPR v2 nickase construction 5 -CACCGATAGGCTG TCCTTTGTCGAC-3 5 -CACCGCCTCGACACCCGATTCAAAG-3