Supplementary Materials

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1 Supplementary Materials Recurrent SMARCA4 Mutations in Small Cell Carcinoma of the Ovary Petar Jelinic, Jennifer J. Mueller, Narciso Olvera, Fanny Dao, Sasinya N. Scott, Ronak Shah, JianJiong Gao, Nikolaus Schultz, Mithat Gonen, Robert A. Soslow, Michael F. Berger, Douglas A. Levine Table of Contents: I. Supplementary Figures Supplementary Figure 1. Sequence analyses for SMARCA4 in SCCOHT cases. Supplementary Figure 2. SMARCA4 gene expression across TCGA tumors. Supplementary Figure 3. Immunohistochemistry (IHC) for SMARCA4 in SCCOHT cases. Supplementary Figure 4. Analysis of homozygous deletion in case 103. Supplementary Figure 5. Analysis of splice site variant in case 102. Supplementary Figure 6. Sequence analyses for SMARCA4 in H1299 cell line. Supplementary Figure 7. SMARCA4 effect on cell proliferation. Supplementary Figure 8. Overall survival among lung adenocarcinoma TCGA. Supplementary Figure 9. Histopathological features of a SCCOHT. II. Supplementary Tables Supplementary Table 1. Summary of patient characteristics and SMARCA4 mutations Supplementary Table 2. Additional somatic mutations. Supplementary Table 3. List of 279 genes screened for mutations. Supplementary Table 4. Summary of immunohistochemistry studies. Supplementary Table 5. Nonsynonymous somatic mutations identified in all genes.

2 #101 G>A #102 G>A #104 G>A #104 C>T Exon 24 Intron 24 Exon 27 Intron 27 Exon 9 Intron 9 Exon 24 Intron 24 Exon 27 Intron Supplementary Figure 1. Sequence analyses for SMARCA4 in SCCOHT cases. Next- genera:on sequence coverage demonstra:ng iden:fied variants (top panels) and valida:on through Sanger sequencing (bodom panels).

3 #105 C>T #105 TC>T #106 TC>T #106 C>T Supplementary Figure 1, continued

4 #107 GACGA GACCGTCA>G #108 C>T del Supplementary Figure 1, continued

5 #109 T>TG #109 G>T #110 C>T Supplementary Figure 1, continued

6 #111 C>T #112 G>A Exon 18 Intron Supplementary Figure 1, continued

7 SMARCA4 expression -- RNA-Seq (RSEM) Missense In frame Nonsense Frameshift Splice Site No Mutation AML Bladder Breast Cervical Colorectal GBM Head & neck Kidney clear cell Kidney chromophobe Kidney papillary Low grade glioma Lung adenoca. Lung squamous Ovary Prostate Pancreas Melanoma Thyroid Supplementary Figure 2. SMARCA4 gene expression across TCGA tumors for cases with available muta:on and RNA- seq data (RSEM). A correla:on is seen between inac:va:ng SMARCA4 muta:ons and decreased gene expression across various solid tumors. A two- sided Student s t test was used to compare samples with non- missense muta:ons and other samples without muta:ons or with only missense muta:ons. For all TCGA samples, the mean RNA- seq RSEM (2,050, s.d. of 1,760) was less in samples with non- missense muta:ons than in other samples without muta:ons or with only missense muta:ons (3,724, s.d. of 1,692; P = ). For TCGA lung adenocarcinoma samples, the mean RNA- seq RSEM (601, s.d. of 370) was less in samples with non- missense muta:ons than in other samples without muta:ons or with only missense muta:ons (3,330, s.d. of 1,524; P = ).

8 Positive control #103 #110 #109 #101 #102 #106 #108 #111 #112 Supplementary Figure 3. Immunohistochemistry for SMARCA4 in SCCOHT cases. High- grade serous ovarian carcinoma is used as a posi:ve control. Case numbers are indicated in each panel. Immunohistochemistry results are provided in Supplementary Table 1. Note the intense staining of blood vessels and stromal cell nuclei as internal controls.

9 B) A) bp ladder 103 Fx/Rx N T 103 Rx 103 Fx Exon 27 Exon 24 C) Up Fx Up Rx 100bp ladder Up Fx/Rx Down Fx/Rx N T N T Down Fx Down Rx Supplementary Figure 4. Analysis of homozygous dele:on in case 103. Next- genera:on sequence coverage demonstra:ng that exons 25 and 26 are deleted. An electropherogram from Sanger sequencing of cdna valida:ng that the dele:on retains an ORF from exon 24 to exon 27 (Panel A). One- step RT- PCR confirms that tumor :ssue yields a single band with primers that span exons 24 and 27 (Panel B;, nonspecific band). One- step RT- PCR with primers targe:ng regions upstream and downstream from the dele:on site show equal expression, demonstra:ng con:nua:on of transcrip:on downstream from the dele:on (Panel C).

10 b Up Fx Up Rx 100bp ladder Up Fx/Rx Down Fx/Rx N T N T Down Fx Down Rx Supplementary Figure 5. Analysis of splice site variant in case 102. One- step RT- PCR confirms that the exon- intron band is preferen:ally expressed over the exon- exon band in tumor :ssue (Panel A). One- step RT- PCR with primers targe:ng regions upstream and downstream from the muta:on site show equal expression, demonstra:ng con:nua:on of transcrip:on downstream from the muta:on (Panel B). Immunoblots are shown in Figure 2b. The exon- exon primers detected weaker bands, reflec:ng loss of expression in tumor :ssues compared with normal :ssues in cases with splice- site muta:ons. The exon- intron primers demonstrated equivalent to greater expression of the retained intron in the tumor :ssues. As SMARCA4 introns may be retained in non- cancer :ssues, some intronic expression is expected in normal :ssues. These data taken together indicate preferen:al intronic expression, as expected, in cdna sequenced from tumor samples with splice- site muta:ons.

11 WT H nt deletion Supplementary Figure 6. Sequence analyses for SMARCA4 in H1299 cell line. An electropherogram from Sanger sequencing of genomic DNA valida:ng a 69- nt dele:on in the ORF of this control cell line that results in loss of protein expression, as shown in Figure 2b.

12 SMARCA4/µg A) B) SMARCA4 p21 β-actin C) D) SMARCA4 β-actin Relative Cell Proliferation Cell Number (x10^6) SMARCA4/µg shntc shsmarca4 24h 48h 72h Supplementary Figure 7. SMARCA4 effect on cell prolifera:on. SMARCA4 overexpression in H1299 cells. Representa:ve immunoblot from three biologic replicates demonstrates a correla:on between increased SMARCA4 and p21 expression (Panel A). Cell growth assessment in H1299 cells overexpressing SMARCA4. Mean cell counts from three biologic replicates (Panel B). Representa:ve immunoblot confirmed SMARCA4 knockdown in 293T cells using shrna. As a control, shntc (Non- Targe:ng Control) was used (Panel C). XTT prolifera:on assay in 293T cells depleted of SMARCA4. Means represent three independent experiments (Panel D).

13 100.0% 90.0% 80.0% 70.0% With nonsense or frameshift mutations in SMARCA4 With only missense or no mutations in SMARCA4 Logrank Test P-Value: Surviving 60.0% 50.0% 40.0% 30.0% 20.0% 10.0% 0.0% Months Survival Supplementary Figure 8. Overall survival among lung adenocarcinoma TCGA cases based on inac:va:ng SMARCA4 muta:ons. Median overall survival was 11.6 months among 6 pa:ents with inac:va:ng SMARCA4 muta:ons compared with 44.6 months for 197 pa:ents without inac:va:ng muta:ons.

14 Supplementary Figure 9. Histopathological features of an SCCOHT. The typical histopathological features of SCCOHT, including a combina:on of small neoplas:c cells forming a pseudofollicular space and larger rhabdoid cells, are visible in a sample obtained from 1 of 12 tumors that were subjected to target capture and massively parallel DNA sequencing (hematoxylin and eosin).

15 Case Number Supplementary Table 1. Summary of patient characteristics and SMARCA4 mutations. Age at diagnosis (years) Year of diagnosis Coding sequence change Predicted protein change Variant class sequence reads allele frequency Affected exon Functional domain # G>A p.q1182_splice Splice site Helicase Loss IHC result G>A p.k1390_splice Splice site Loss homozygous deletion Exon deletion NA NA Helicase Retained G>A p.k587_splice Splice site Not available C>T p.r978 Nonsense SNF C>T p.q331 Nonsense Not available Frameshift TC>T p.i542fs deletion C>T p.r1093 Nonsense Loss Frameshift TC>T p.l388fs deletion Helicase GACGAGACCGT CA>G p.etvn1300del In frame deletion Not available C>T p.q847 Nonsense SNF2 Loss T>TG p.l762fs Frameshift insertion SNF2 Loss G>T p.g836 Nonsense SNF C>T p.q1166 Nonsense Helicase Loss Nonsense C>T p.r1005 germline^ SNF2 Loss G>A p.k953_splice Splice site SNF2 Equivocal # See Figure 1 ^ Germline nonsense mutation with somatic loss of heterozygosity IHC immunohistochemistry; NA - not applicable

16 Supplementary Table 2. Additional somatic mutations identified in SCCOHT patients. Case Number Age at diagnosis (years) Year of Diagnosis Gene Coding Sequence Change Predicted Protein Change Variant Class sequence reads allele frequency JAK3 G>A p.s147n Missense ASXL1 C>CG p.s147n Frameshift insertion Frameshift NOTCH2 CT>T p.s1855fs deletion WT1 A>C p.k414q Missense

17 Supplementary Table 3. List of 279 genes screened for mutations. ABL1 ABL2 AKT1 AKT2 AKT3 ALK ALOX12B APC AR ARAF ARHGAP26 ARID1A ARID2 ASXL1 ATM ATRX AURKA BAP1 BCL2L1 BCL2L11 BCL6 BCOR BIRC2 BRAF BRCA1 BRCA2 BUB1B CARD11 CBL CBLB CBLC CC1 CCNE1 CD79B CDC42EP2 CDC73 CDH1 CDH11 CDK12 CDK4 CDK6 CDK8 CDKN1A CDKN2A CDKN2B CDKN2C CEBPA CHEK1 CHEK2 CIC CREBBP CRKL CRLF2 CSF1R CTNNB1 CYLD DAXX DDR2 DICER1 DIS3 DNMT1 DNMT3A DNMT3B E2F3 EGFR EIF4EBP1 EP300 EPHA10 EPHA2 EPHA3 EPHA4 EPHA5 EPHA6 EPHA7 EPHA8 EPHB1 EPHB2 EPHB3 EPHB4 EPHB6 ERBB2 ERBB3 ERBB4 ERG ESR1 ETV1 ETV6 EZH2 FAM123B FAM46C FAS FAT1 FBXO11 FBXW7 FGFR1 FGFR2 FGFR3 FGFR4 FH FLCN FLT1 FLT3 FLT4 FOXL2 FUBP1 GATA1 GATA2 GATA3 GLI1 GLI3 GNA11 GNAQ GNAS GOLPH3 GRIN2A GRM3 GSK3B HDAC2 HIF1A HMGA2 HNF1A HRAS HSP90AA1 IDH1 IDH2 IGF1R IGFBP7 IKBKE IKZF1 IL7R INPP4A INPP4B INSR IRS1 IRS2 JAK1 JAK2 JAK3 JUN KCNJ5 KDM5C KDM6A KDR KEAP1 KIT KLF6 KRAS LDHA LGR6 LMO1 MAGI2 MAP2K1 MAP2K2 MAP2K4 MAP3K1 MAP3K8 MCL1 MDM2 MDM4 MED12 MEF2B MEN1 MET MITF MLH1 MLL MLL2 MLL3 MLST8 MPL MSH2 MSH6 MTOR MYB MYC MYCL1 MYCN MYD88 NCOA2 NF1 NF2 NFE2L2 NFKB1 NFKB2 NKX2-1 NOTCH1 NOTCH2 NOTCH3 NOTCH4 NPM1 NRAS NTRK1 NTRK2 NTRK3 PAK7 PALB2 PARK2 PARP1 PAX5 PBRM1 PDGFRA PDGFRB PHOX2B PIK3C2G PIK3CA PIK3CB PIK3CD PIK3CG PIK3R1 PIK3R2 PIK3R3 PKM2 PLK2 PNRC1 PPP2R1A PRDM1 PREX2 PRKAA2 PRKAR1A PRKCI PTCH1 PTEN PTPN11 PTPRD PTPRS PTPRT RAF1 RARA RB1 REL RET RICTOR RNF43 ROR2 ROS1 RPTOR RUNX1 SDHB SETD2 SF3B1 SHQ1 SMAD2 SMAD3 SMAD4 SMARCA4 SMARCB1 SMO SOCS1 SOX2 SPOP SRC SRSF2 STAG2 STK11 SUFU TBK1 TEK TERT TET1 TET2 TGFBR2 TMPRSS2 TNFAIP3 TNFRSF14 TOP1 TP53 TP63 TSC1 TSC2 TSHR U2AF1 VHL WAS WNK1 WT1 XPO1 YAP1 YES1 ZRSR2

18 Supplementary Table 4. Summary of immunohistochemistry studies performed to assist with diagnostic interpretation. Case EMA P53 Cytokeratin Inhibin LCA CD10 S100 Desmin WT = not done

19 Supplementary Table 5. Nonsynonymous somatic mutations identified in all genes. Patient Chrom Start Ref Alt Hugo_Symbol Variant_Class Protein_Change Codon_Change T_TotalDepth T_AltFreq G A SMARCA4 Splice_Site p.q1182_splice c.3546_splice G A SMARCA4 Splice_Site p.k1390_splice c.4170_splice G A SMARCA4 Splice_Site p.k587_splice c.1761_splice C T SMARCA4 Nonsense_Mutation p.r978 c.2932c>t C T SMARCA4 Nonsense_Mutation p.q331 c.991c>t TC T SMARCA4 Frame_Shift_Del p.i542fs c.1625_1626tc>t TC T SMARCA4 Frame_Shift_Del p.l388fs c.1164_1165tc>t C T SMARCA4 Nonsense_Mutation p.r1093 c.3277c>t GACGAG c.3895_3907gac ACCGTCA G SMARCA4 In_Frame_Del p.etvn1300del GAGACCGTCA>G C T SMARCA4 Nonsense_Mutation p.q847 c.2539c>t T TG SMARCA4 Frame_Shift_Ins p.l762fs c.2285_2285t>tg G T SMARCA4 Nonsense_Mutation p.g836 c.2506g>t C T JAK3 Missense_Mutation p.s147n c.440g>a C CG ASXL1 Frame_Shift_Ins p.r397fs c.1189_1189c>cg AG A NOTCH2 Frame_Shift_Del p.s1855fs c.5564_5565ct>t C T SMARCA4 Nonsense_Mutation p.q1166 c.3496c>t Germline LOH / C T SMARCA4 Nonsense p.r1005 c.3013c>t T G WT1 Missense_Mutation p.k414q c.1240a>c G A SMARCA4 Splice_Site p.k953_splice c.2859_splice