Supplementary Table S1. Histopathologic findings for multi-region analysis.
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1 SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLES Supplementary Table S1. Histopathologic findings for multi-region analysis. Patient Histology R1: clear cell RCC, Fuhrman 2-3, acinar pattern 1 R2: clear cell RCC, Fuhrman 3-4, large alveolar pattern R3: clear cell RCC, Fuhrman 3-4, large alveolar pattern R1: clear cell RCC, Fuhrman 3, large alveolar pattern R2: clear cell RCC, Fuhrman 3-4, large alveolar pattern 2 R3: clear cell RCC, Fuhrman 3-4, large alveolar pattern M1: clear cell RCC, Fuhrman 3-4, large alveolar pattern R1: clear cell RCC, Fuhrman 2-3, acinar and tubulopapillary patterns R2: clear cell RCC, Fuhrman 3-4, large alveolar and solid patterns 3 R3: clear cell RCC, Fuhrman 3-4, solid pattern R4: clear cell RCC, Fuhrman 3-4, solid pattern R1: clear cell RCC, Fuhrman 2, acinar pattern 4 M1: clear cell RCC, Fuhrman 2, acinar pattern 5 R1: Unclassified RCC, intermixed clear and eosinophilic cells, Fuhrman 2-3, papillary architecture 1
2 Supplementary Table S2. IMPACT gene list and their respective chromosomal positions. Gene Symbol RefSeq ID Chromosome ABL1 NM_ q34.1 ABL2 NM_ q25.2 AKT1 NM_ q32.32-q32.33 AKT2 NM_ q13.1-q13.2 AKT3 NM_ q44 ALK NM_ p23 ALOX12B NM_ p13.1 APC NM_ q21-q22 AR NM_ Xq12 ARAF NM_ Xp11.3-p11.23 ARHGAP26 NM_ q31 ARID1A NM_ p36.1-p35 ASXL1 NM_ q11 ATM NM_ q22-q23 ATRX NM_ Xq21.1 AURKA NM_ q13 BAP1 NM_ p21.31-p21.2 BCL2L1 NM_ q11.21 BCL6 NM_ q27 BIRC2 NM_ q22 BRAF NM_ q34 BRCA1 NM_ q21-q24 BRCA2 NM_ q12-q13 CARD11 NM_ p22 CBL NM_ q23.3-qter CBLB NM_ q CBLC NM_ q13.2 CCND1 NM_ q13 CCNE1 NM_ q12 CD79B NM_ q23 CDC42EP2 NM_ q13 CDC73 NM_ q25 CDH1 NM_ q22.1 CDK4 NM_ q13 CDK6 NM_ q21-q22 CDK8 NM_ q12 CDKN2A NM_ p21 CDKN2B NM_ p21 CDKN2C NM_ p32.3 CEBPA NM_ q13.1 CHEK1 NM_ q24.2 CHEK2 NM_ q12.1 2
3 CREBBP NM_ p13.3 CRKL NM_ q11.21 CRLF2 NM_ Xp22.3 and Yp11.3 CSF1R NM_ q32 CTNNB1 NM_ p21 CYLD NM_ q12-q13 DAXX NM_ p21.3 DDR2 NM_ q12-q23 DICER1 NM_ q32.2 DIS3 NM_ q21.32 DNMT1 NM_ p13.2 DNMT3A NM_ p23 DNMT3B NM_ q11.2 EGFR NM_ p12 EIF4EBP1 NM_ p12 EP300 NM_ q13.2 EPHA3 NM_ p11.2 EPHA5 NM_ q13.1 EPHA6 NM_ q12.1 EPHA7 NM_ q16.3 EPHA8 NM_ p36.12 EPHB1 NM_ q21-q23 EPHB4 NM_ q22 EPHB6 NM_ q33-q35 ERBB2 NM_ q11.2-q12 ERBB3 NM_ q13 ERBB4 NM_ q33.3-q34 ERG NM_ q22.3 ESR1 NM_ q24-q27 ETV1 NM_ p22 ETV6 NM_ p13 EZH2 NM_ q35-q36 FAM123B NM_ Xq11.1 FAM46C NM_ p12 FAS NM_ q24.1 FBXW7 NM_ q31.23 FGFR1 NM_ p12 FGFR2 NM_ q25.3-q26 FGFR3 NM_ p16.3 FGFR4 NM_ q33-qter FH NM_ q42.1 FLCN NM_ p11.2 FLT1 NM_ q12 FLT3 NM_ q12 FOXL2 NM_ q23 GATA1 NM_ Xp11.23 GATA2 NM_ q21 GATA3 NM_ p15 3
4 GNA11 NM_ p13.3 GNAQ NM_ q21 GNAS NM_ q13.2-q13.3 GOLPH3 NM_ p13.2 GRIN2A NM_ p13.2 GSK3B NM_ q13.3 HDAC2 NM_ q21 HIF1A NM_ q23.2 HMGA2 NM_ q15 HNF1A NM_ q24.31 HRAS NM_ p15.5 HSP90AA1 NM_ q32.33 IDH1 NM_ q32-qter IDH2 NM_ q21-qter IGF1R NM_ q26.3 IGFBP7 NM_ q12 IKBKE NM_ q31 IKZF1 NM_ pter-7qter INSR NM_ p13.3-p13.2 IRS1 NM_ q36 IRS2 NM_ q34 JAK1 NM_ p32.3-p31.3 JAK2 NM_ p24 JAK3 NM_ p13-p12 JUN NM_ p32-p31 KDM5C NM_ Xp11.22-p11.21 KDM6A NM_ Xp11.2 KDR NM_ q11-q12 KEAP1 NM_ p13.2 KIT NM_ q11-q12 KLF6 NM_ p15 KRAS NM_ p12.1 LDHA NM_ p15.1 LGR6 NM_ q32.1 MAGI2 NM_ q21 MAP2K1 NM_ q22.1-q22.33 MAP2K2 NM_ p13.3 MAP2K4 NM_ p11.2 MAP3K8 NM_ p11.2 MCL1 NM_ q21 MDM2 NM_ q13-q14 MDM4 NM_ q32 MEN1 NM_ q13 MET NM_ q31 MITF NM_ p14.1-p12.3 MLH1 NM_ p22.3 MLL NM_ q23 MLL2 NM_ q12-q13 4
5 MLL3 NM_ q36 MLST8 NM_ p13.3 MPL NM_ p34 MSH2 NM_ p21 MSH6 NM_ p16 mtor NM_ p36 MYB NM_ q22-q23 MYC NM_ q24 MYCL1 NM_ p34.3 MYCN NM_ p24.3 NCOA2 NM_ q13 NF1 NM_ q11.2 NF2 NM_ q12.2 NFE2L2 NM_ q31 NFKB1 NM_ q24 NFKB2 NM_ q24 NKX2-1 NM_ q13.3 NOTCH1 NM_ q34.3 NOTCH2 NM_ p13-p11 NOTCH3 NM_ p13.2-p13.1 NOTCH4 NM_ p21.3 NPM1 NM_ q35.1 NRAS NM_ p13.2 NTRK1 NM_ q21-q22 NTRK2 NM_ q22.1 NTRK3 NM_ q24-q25 PAK7 NM_ p12 PARK2 NM_ q25.2-q27 PARP1 NM_ q41-q42 PAX5 NM_ p13.2 PBRM1 NM_ p21 PDGFRA NM_ q12 PDGFRB NM_ q33.1 PHOX2B NM_ p13 PIK3C2G NM_ p12 PIK3CA NM_ q26.3 PIK3CB NM_ q21-qter PIK3CD NM_ p36.2 PIK3CG NM_ q22 PIK3R1 NM_ q13.1 PIK3R2 NM_ q13.2-q13.4 PIK3R3 NM_ p34.1 PKM2 NM_ q22-qter PLK2 NM_ q12.1-q13.2 PNRC1 NM_ q16.1 PREX2 NM_ q13.1 PRKAR1A NM_ q23-q24 PRKCI NM_ q26.3 5
6 PTCH1 NM_ q22.1-q31 PTEN NM_ q23 PTPN11 NM_ q24.1 PTPRD NM_ p24.1-p23 PTPRS NM_ p13.3 RAF1 NM_ p25 RARA NM_ q21.1 RB1 NM_ q14.2 REL NM_ p13-p12 RET NM_ q11.2 RICTOR NM_ p13.1 RPTOR NM_ q25.3 RUNX1 NM_ q22.3 SDHB NM_ p36.1-p35 SETD2 NM_ p21.31 SHQ1 NM_ p13 SMAD4 NM_ q21.1 SMARCA4 NM_ p13.3 SMARCB1 NM_ q11.23 SMO NM_ q32.1 SOCS1 NM_ p13.13 SOX2 NM_ q26.3-q27 SPOP NM_ q21.33 SRC NM_ q12-q13 STK11 NM_ p13.3 SUFU NM_ q24.32 TBK1 NM_ q14.2 TEK NM_ p21 TERT NM_ p15.33 TET1 NM_ q21 TET2 NM_ q24 TGFBR2 NM_ p22 TMPRSS2 NM_ q22.3 TNFAIP3 NM_ q23-q25 TOP1 NM_ q12-q13.1 TP53 NM_ p13.1 TP63 NM_ q27-q29 TSC1 NM_ q34 TSC2 NM_ p13.3 TSHR NM_ q24-q31 VHL NM_ p25.3 WT1 NM_ p13 YAP1 NM_ q13 YES1 NM_ p11.31-p
7 Supplementary Table S3. Impact assay run statistics on all samples. Please separate excel spreadsheet, table too large for inclusion here. Supplementary Table S4. Impact assay exon coverage on all samples. Please separate excel spreadsheet, table too large for inclusion here. 7
8 Supplementary Table S5. WEC run statistics on patients #4 and #5. PF UQ BASES ALIGNED PCT SELECTED BASES MEAN TARGET COVERAGE PCT USABLE BASES ON TARGET PCT TARGET BASES 2X PCT TARGET BASES 10X PCT TARGET BASES 20X PCT TARGET BASES 30X TOTAL SAMPLE READS Pt 4 (N) 70,182,339 5,123,974, % % 96.54% 91.96% 86.08% 79.47% Pt 4 (T) 54,314,779 3,920,678, % % 95.86% 89.77% 81.73% 72.13% Pt 5 (N) 77,253,432 5,659,907, % % 96.39% 91.70% 86.10% 80.08% Pt 5 (T) 93,018,700 6,812,455, % % 96.67% 92.90% 88.33% 83.49% 8
9 Supplementary Table S6. List of all non-variant mutations detected by IMPACT assays in individual patient samples. Gene Sample Chr Genomic Coordinates (GRCh37) REF ALT AA Change Effect Transcript ID Allele Freq % VHL Pt G T E94* Nonsense NM_ PBRM1 Pt T A E991D Missense NM_ PHOX2B Pt C A G20V Missense NM_ NFKB1 Pt T C L611P Missense NM_ NFKB1 Pt C T A623V Missense NM_ TSC1 Pt G - P311fs Frameshift NM_ VHL Pt C A H115N Missense NM_ TP53 Pt C T R273H Missense NM_ JAK1 Pt C - R110fs Frameshift NM_ IGF1R Pt C S847fs Frameshift NM_ BAP1 Pt C A Splice e9-1 Splice Site NM_ VHL Pt GA - G212 Frameshift NM_ MTOR Pt G T Q2223K Missense NM_ MLL3 Pt A G V2060A Missense NM_ VHL Pt T C L118P Missense NM_ PBRM1 Pt TCACTGCTGAA - E1360fs Frameshift NM_ ATM Pt T - N1044fs Frameshift NM_ DAXX Pt G A T684M Missense NM_ KEAP1 Pt G A S102L Missense NM_
10 Supplementary Table S7. Non-variant mutations identified by WEC in patients #4 and #5. Genomic Coordinates (GRCh37) REF ALT AA Gene Pt ID Chr Change Effect Transcript.ID AKR7A3 Pt A T M/K Missense NM_ SLC35A3 Pt C T L/F Missense NM_ TROVE2 Pt GA G. Frameshift NR_ CAD Pt C G S/R Missense NM_ OXER1 Pt T A H/L Missense NM_ RANBP2 Pt A T N/Y Missense NM_ ZNF717 Pt TC T. Frameshift NM_ ATP6V1G2- DDX39B Pt T C K/E Missense NR_ ABCF1 Pt T C F/S Missense NM_ ALDH8A1 Pt T C S/G Missense NM_ JARID2 Pt GA G. Frameshift NM_ NEUROD6 Pt G A P/S Missense NM_ TOPORS Pt T C N/S Missense NM_ HABP4 Pt G A A/T Missense NM_ PBLD Pt A T I/N Missense NM_ FAM171A1 Pt TG T. Frameshift NM_ ATM Pt AT A. Frameshift NM_ KLF5 Pt G T W/L Missense NM_ ANKRD20A9P Pt C CA. Frameshift NR_ MIR1197 Pt GA G. Frameshift NR_ PLA2G15 Pt T G L/W Missense NM_ ITGA3 Pt C A P/Q Missense NM_ TMX4 Pt G A R/W Missense NM_ C20orf118 Pt T A F/I Missense NM_ TSHZ2 Pt A T K/* Nonsense NM_ KIF17 Pt C A R/M Missense NM_ AGL Pt A G K/E Missense NM_ IGSF8 Pt G T A/E Missense NM_ PRG4 Pt A T R/* Nonsense NM_ FBXO2 Pt C CGCG A/AP Frameshift NM_ WDR54 Pt A G S/G Missense NM_ STAMBP Pt CT C. Frameshift NM_ PVRL3 Pt G A W/* Nonsense NM_ ISY1 Pt C G. Splice Site NM_ ISY1-RAB43 Pt G T L/I Missense NM_ C3orf25 Pt T C K/E Missense NM_ SI Pt A C V/G Missense NM_ COL7A1 Pt CT C. Frameshift NM_ DCP1A Pt TA T. Frameshift NM_ PARP14 Pt T TAC. Frameshift NM_ Allele Freq % 10
11 PPEF2 Pt G T P/H Missense NM_ DAB2 Pt T C K/E Missense NM_ SSBP2 Pt T C R/G Missense NM_ NMUR2 Pt C T C/Y Missense NM_ TAP1 Pt G A P/S Missense NM_ DAXX Pt G A R/* Nonsense NR_ FTSJD2 Pt G A E/K Missense NM_ STL Pt A T Y/N Missense NR_ GTPBP10 Pt A C E/A Missense NM_ SSPO Pt GC G. Frameshift NM_ VCPIP1 Pt T A N/I Missense NM_ TJP2 Pt A T T/S Missense NM_ ODF2 Pt A G E/G Missense NM_ NOXA1 Pt G C G/R Missense NM_ GAD2 Pt G A G/S Missense NM_ ZNF33A Pt A G I/V Missense NM_ MCU Pt T C Y/H Missense NM_ P4HA1 Pt T C N/S Missense NM_ KIAA0913 Pt T C V/A Missense NM_ ECHS1 Pt T G K/T Missense NM_ AGAP4 Pt CA C. Frameshift NM_ IFIT5 Pt G GT. Frameshift NM_ NAP1L4 Pt T G E/D Missense NM_ SPON1 Pt C G P/R Missense NM_ SLC22A24 Pt A G F/S Missense NM_ MALAT1 Pt A T K/N Missense NR_ MALAT1 Pt A T I/F Missense NR_ FAM138D Pt GT G. Frameshift NR_ ATP8A2 Pt A G K/R Missense NM_ ANKRD20A9P Pt C CA. Frameshift NR_ MIS18BP1 Pt C G D/H Missense NM_ NEMF Pt G T S/* Nonsense NM_ TDP1 Pt G A R/Q Missense NM_ SAV1 Pt GA G. Frameshift NM_ SPATA5L1 Pt A G T/A Missense NM_ SMYD4 Pt T A R/* Nonsense NM_ FLJ90757 Pt C A R/M Missense NR_ C19orf28 Pt A G L/P Missense NM_ KEAP1 Pt G A S/L Missense NM_ KLK9 Pt G A L/F Missense NM_ NLRP12 Pt G C T/R Missense NM_ SLC9A8 Pt C A P/T Missense NM_ SON Pt G A R/Q Missense NM_ PI4KA Pt G A R/* Nonsense NM_ POM121L8P Pt AC A. Frameshift NR_ TLR8 Pt 5 X T G F/V Missense NM_ CYBB Pt 5 X G T. Splice Site NM_
12 SUPPLEMENTARY FIGURES Supplementary Figure 1. Flow chart depicts the IMPACT assay mutation identification and filtering algorithm. Images Intensities Base Calls and Qualities Fastq Conversion, Demultiplexing Alignment Re-Alignment (Local MSA) Remove Duplicates Recalibrate Base Qualities Performance Metrics Alignment Rate Coverage Distribution On-Target Specificity Library Complexity Sequence Variants SNVs Structural Variants Cancer Variants Somatic Mutations Indels Copy Alterations Rearrangements 12
13 Supplementary Figure 2. Flow chart depicts the WEC assay mutation identification and filtering algorithm. Remove Adapters Align BWA (hg19) Resolve Overlaps Mark Duplicates (Picard) Remove Multi-Mapper GATK Realignment Annotations dbsnp SNP eff Intersection of Mu Tect /GATK Germline Low Qual <3x in Tumor GATK BaseQ (Recalibration) Mu Tect/GATK Unified Genotyper VCF (Total Mutations) VQSR (Recalibrate VCF) Annotated VCF MAF Mark Potential Errors Near Indels SNP Cluster Low Qual 13
14 Supplementary Figure 3. Sanger validations of mutations in the targeted pathway indentified by IMPACT assay for R1 of patients #1,#2, and #3. 14
15 Supplementary Figure 4. Sanger validations of mutations in the targeted pathway indentified by IMPACT assay for additional regions analyzed for patients #1, and #3. Nucleotide changes are circled in red. 15
16 Supplementary Figure 5. The Q2223K mtor mutation provokes hyperactivation of mtorc1 in serum free growth conditions. Immunoblot showing that a higher level of phosphorylation of S6K (T389) induced by Q2223K compared to wild-type mtor can be observed for hours following initiation of serum deprivation. HEK293T cells were co-transfected like in Figure 3d, and 24 hours later, cells were washed and subsequently maintained in serum free medium for the indicated lengths of time (1 to 20 hours) before harvest. 16
17 Supplementary Figure 6. Hyperactivation of mtorc1 induced by Q2223K is sensitive to rapalog-based mtor inhibition. Immunoblot showing that phosphorylation of S6K (T389), promoted by wild-type and Q2223K mtor, is markedly reduced by everolimus, temsirolimus, and rapamycin. Like in Figure 3e, HEK293T cells were co-transfected with Myc-tagged S6K and an expression plasmid for wild-type or Q2223K mtor. 24 hours later, cells were washed with serum free medium and exposed to 10% serum for 1 hour in the absence (-) or presence of the indicated concentrations of rapalog. 17
18 Supplementary Figure 7. Immunoblots detecting phosphorylated mtor at serine residues 2448 (S2448) and 2481 (S2481). Compared to wild-type mtor, the Q2223K mutant shows elevated phosphorylation at mtor (S2448), but not at (S2481). HEK293T cells were co-transfected and treated like in Figure 3d. 18
19 Supplementary Figure 8. Copy number plots for multiple tumor regions in patient #3 showing the loss of chromosome 9 only in tumor regions carrying the TSC1 nonsense mutation (R3, R4). R1 R2 R3 9- R chr 19
20 Supplementary Figure 9. Copy number plots for patient with tuberous sclerosis complex and metastatic unclassified RCC with extended benefit from everolimus therapy (44+ months). Plots show inherited hemizygous of TSC2 in the germline DNA and the current somatic loss (homozygous deletion) in the tumor 1 (T1), while tumor 2 (T2) had the inherited TSC2 loss with a concomitant nonsense mutation in the other allele (Q794*) shown in Sanger validation. N (vs unmatched N) - Germline T1 (vs unmatched N) 16p (-/+) 16p (-/-) 20
21 Supplementary Figure 10. PI3K pathway alterations as reported across 418 cases of clear cell RCC in TCGA. Alterations are detected in 42% of cases. Queried through the cbio cancer genomics portal (Cerami et al., Cancer Discov May;2(5): doi: / CD ). 21
22 Supplementary Figure 11: Hematoxylin and eosin (H&E) stains and immunohistochemistry for 4EBP1 phosphorylation (Thr 37/46), an mtorc1 downstream effector, in primary kidney tumors and adjacent normal kidney tissue for patients
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