Targetable Alterations in Adult Patients with Soft-Tissue Sarcomas

Supplementary Online Content

Lucchesi C, Khalifa E, Laizet Y, et al. Genomic landscape of adult soft-tissue sarcomas: moving toward personalized medicine [published online May 3, 2018] . JAMA Oncol. doi:10.1001/jamaoncol.2018.0723

eMethods. eTable 1. Actionable alterations in a series of 584 STS eTable 2. Clinical outcome of STS patients treated with targeted therapies eFigure 1. Proportion of genetic alterations according to genetic class of STS eFigure 2. Distribution of the number of alterations eFigure 3. Lollipop-style mutation diagrams for frequently altered genes eFigure 4. Distribution of actionable alterations in the 10 most frequent histological subtypes of STS eFigure 5. Co-occurrence and mutual exclusivity of genetic alterations in STS eFigure 6. Objective responses to targeted therapies in STS eFigure 7. Design of the MULTISARC study

This supplementary material has been provided by the authors to give readers additional information about their work.

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SUPPLEMENTARY METHODS

Bioinformatics Analysis

Percentage of Sarcoma samples covered by the different NGS assays of AACR Genie

The GENIE data guide (referenced in the Material and Methods: http://www.aacr.org/Documents/GENIEDataGuide.pdf) describes the characteristics of NGS assays used in the study. 584 adults Soft tissue Sarcoma have been sequenced with differently-sized NGS assays whose cumulative sample distribution is reported below. It shows that 493 samples (84%, n=584) are covered by the 4 large-panel NGS assays of the MSK and DFCI Institutes, which include from 300 to 451 genes.

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Data mining of the oncoKB data portal Targetable alterations from the oncoKB portal (http://oncokb.org) were selected via a manual process curated and supervised by our Clinical Department. The alterations and their annotations were extracted in a text table containing the gene symbol, the exact or generic alteration details and the oncoKB therapeutic classification. Then the GENIE data tables data_clinical.txt (patient annotation data), data_CNA.txt (copy number), data_fusions.txt (translocations) and data_mutations_extended.txt (snv/indel) (source https://www.synapse.org/#!Synapse:syn7844527) were filtered to include only the patient covered by the study and automatically mined for each of the following alteration scenarios:

a) Explicit mutations, for which both gene and exact genetic alteration nomenclature was known (e.g. BRAF V600E): the columns and of GENIE data_mutations_extended.txt table was looked for and the exact match of gene and alteration was reported

b) Functional mutations, for which the gene was known and but only the mutation type: the columns and of GENIE data_mutations_extended.txt table were looked for the code “missense_variant” for oncogenic mutations and the code list "frameshift_variant","frameshift_variant,splice_region_variant" for truncating mutations., "inframe_deletion,splice_region_variant","missense_variant,splice_region_variant","protein _altering_variant","splice_acceptor_variant","splice_acceptor_variant,coding_sequence_vari ant","splice_acceptor_variant,coding_sequence_variant,intron_variant","splice_acceptor_va riant,intron_variant","splice_donor_variant","splice_donor_variant,coding_sequence_varian t","splice_donor_variant,coding_sequence_variant,intron_variant","splice_region_variant,int ron_variant","splice_region_variant,synonymous_variant","start_lost","stop_gained","stop_ gained,protein_altering_variant","stop_gained,splice_region_variant","stop_lost" for inactivating mutations.

c) Genomic amplifications (e.g. CDK4 amplifications) and homozygous deletions (e.g. SMARCB1 deletion): the GENIE data_CNA.txt table was looked for couples patient/gene whose CAN value was +2 for “amplifications” or -2 for homozygous deletion.

d) Explicit gene fusions (e.g. KIAA1549-BRAF fusion) : the GENIE data_fusions.txt table was looked for matches of gene1 – gene2 as well as gene2-gene1 gene fusions

e) Gene fusions with only one partner known (e.g. FGFR2 fusions): the GENIE data_fusions.txt table was looked for matches of gene1 – geneX where geneX was a generic partner

When the match was positive a count of 1 was reported for the alteration in the patient. All data analysis have been performed using basic R and Dplyr libraries.

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Supplementary Table 1: Actionable alterations in a series of 584 STS

Supplementary Table 1. Actionable alterations in a series of 584 STS Targetable Genomic sarcomes genomics Complex sarcomas - Translocation Other Total Alteration

sarcomas

related related

Standard care biomarker predictive of response to drugs not approved for management of sarcoma patients PDGFRA fusion 1 0 0 1 ALK fusions 0 0 0 0 BRAF mutation (V600D, 0 0 0 0 V660E, V600G, V600K, V600M,V600R) BRCA1 truncating 2 0 0 2 mutations BRCA2 truncating 2 0 0 2 mutations CDK4 amplification 8 0 76 84 KIT mutation 3 0 0 3 MET amplification 4 1 1 6 MET mutation 4 0 0 4 PDGFRA mutation 1 0 0 1 RET fusion 0 0 0 0 TSC1 truncating mutations 1 0 0 1 TSC2 truncating mutations 3 0 0 3 Alterations associated with compelling clinical evidence of predictive value but neither biomarker nor drug are standard of care AKT1 mutation (E17K) 0 0 0 0 ALK mutation (L1196M, 0 0 0 0 L1196Q) ARAF mutation (S214A, 0 0 0 0 S214C) BRAF fusion 0 0 0 0 BRAF mutation (K601E, 0 0 0 0 L597Q, L597R, L597S, L597V) ERBB2 amplification 0 0 0 0 ERBB2 oncogenic 5 1 2 8 mutations

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ERCC2 truncating 1 1 0 2 mutations ESR1 oncogenic mutations 2 0 0 2 FGFR1 amplification 3 1 3 7 FGFR2 fusion 0 1 0 1 FGFR3 fusion 0 0 0 0 FGFR3 mutation 2 1 0 3 FLT3 mutation 0 0 0 0 (Y572_Y630ins) IDH1 mutation (R132C, 0 0 0 0 R132G, R132H, R132Q, R132S) IDH2 mutation (R140Q, 0 0 0 0 R172G, R172K, R172M, R172S) JAK2 (PCM1-JAK2 Fusion) 0 0 0 0 MAP2K1 oncogenic 0 0 0 0 mutations MDM2 amplification 16 1 84 101 MET mutations 0 0 0 0 (963_D1010splice, 981_1028splice, X1006_splice, X1007_splice, X1008_splice, X1009_splice, X1010_splice, X963_splice, D1010H, D1010N, D1010Y) MTOR mutation (E2014K) 0 0 0 0 NRAS oncogenic mutations 8 0 0 8 NTRK1 fusions 0 0 0 0 NTRK2 fusions 0 0 0 0 NTRK3 fusions 0 0 0 0 PIK3CA oncogenic 10 5 0 15 mutations PTCH1 truncating 1 0 0 1 mutations ROS1 mutation (D2033N) 0 0 0 0 Alterations associated with compelling biological evidence of predictive value but neither biomarker nor drug are standard of care ALK mutation (R1275Q) 0 0 0 0 ATM mutation (N2875K, 0 0 0 0 R3008C) ATM truncating mutation 5 0 0 5 BRAF mutation (D594E, 0 0 0 0 D594N, G466V, G469A, G469V, G596C, L597Q, L597V)

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BRAF (KIAA1549-BRAF 0 0 0 0 Fusion) CDKN2A truncating 7 0 0 7 mutations EGFR mutations 0 0 0 0 (729_761del, 729_761ins, L858R, 762_823ins, G719A, L861R, S768I) ERBB2 mutations 0 0 0 0 (E770_K831indel, E770_K831ins) ESR1 mutations (D538G, 0 0 0 0 Y537S) EZH2 oncogenic mutations 2 0 0 2 FGFR1 (BCR-FGFR1 Fusion) 0 0 0 0 FGFR3 mutations (G370C, 0 0 0 0 G380R, K650E, K650M, K650N, K650Q, K650R, K650T, R248C, S249C, S371C, Y373C) IDH1 mutations (R132C, 0 0 0 0 R132G, R132H, R132Q, R132S) KRAS oncogenic mutations 4 1 0 5 MTOR mutations (C1483F, 0 0 0 0 F1888L, L2230V, S2215F, T1977K) NF1 truncating mutations 13 1 1 15 PTEN truncating mutations 7 1 0 8 RAF1 mutation (S257L) 0 0 0 0 TSC1 deletions 0 0 0 0 TSC2 deletions 3 0 0 3 PTCH1 deletions 0 0 0 0 KDR (VEFGR2) oncogenic 7 0 1 8 mutations KDR (VEFGR2) 6 0 2 8 amplifications ATM truncating mutations 0 0 0 0 ATM deletions 1 0 0 1 CDKN2A deletions 21 2 3 26 SMARCB1 truncating 0 0 1 1 mutations SMARCB1 deletions 0 1 1 2 MAP2K1 amplifications 0 0 0 0 VHL oncogenic mutations 1 0 0 1 VHL deletions 0 0 0 0 ARAF oncogenic mutations 1 0 0 1 NF2 truncating mutations 3 0 1 4

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NF2 deletions 2 1 0 3 FGF3 amplifications 0 0 1 1 FGF4 amplifications 0 0 1 1 PTEN deletions 9 2 0 11 RICTOR amplifications 2 0 2 4 NF1 deletions 4 0 0 4

Targetable mutation 95 11 6 112 counts Targetable copy number 79 9 174 262 alterations Targetable translocation 1 1 0 2

Number of patients (with 131 19 89 239 at least 1 druggable (40%) (13%) (82%) (41%) alteration)

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Supplementary Table 2: Clinical outcome of STS patients treated with targeted therapies

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Supplementary Figure 1: Proportion of genetic alterations according to genetic class of STS

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Supplementary Figure 2: Distribution of the number of alterations

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Supplementary Figure 3. Lollipop-style mutation diagrams for frequently altered genes

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Supplementary Figure 4: Incidence of the 20 most frequent targetable alterations in a series of 584 STS.

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Supplementary Figure 5: Co-occurrence and mutual exclusivity of genetic alterations in STS

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The analysis of the matrix allows the identification of 9 subsets of samples coded from A to H, having the following characteristics (goodness of fit significance test):

Category A: 57 samples characterized by co-occurrence of MDM2 and CDK4 amplifications plus at least 1 to 6 additional alterations in TP53, ATRX, TERT, CDKN2A, ERBB4 and TNFAIP3 genes (amongst the more frequent). They are significantly enriched in Dedifferentiated Liposarcoma (67%, n=38) followed by Well-Differentiated Liposarcoma (21%, n=12), Intimal Sarcoma (7%,n=4), Sarcoma NOS (4%,n=2) and Liposarcoma (2%,n=1).

Category B: 24 samples characterized by exclusive co-occurrence of the MDM2 and CDK4 amplifications. They are significantly enriched in Dedifferentiated Liposarcoma (46%,n=11) and Well- Differentiated Liposarcoma (46%,n=11) followed by Undifferentiated Pleomorphic Sarcoma (4%,n=1) and Fibrosarcoma (4%,n=1).

Category C: 20 samples having co-occurrence of MDM2 amplifications plus 1 to 3 additional alteration in TERT, PDGFRA, JUN, KDR, KIT, NTRK1, SDHC, DDR2, FGFR1, CARD11, BIRC3, RB1, CDKN2A, ERBB4, KMT2D, PTEN, ROS1 and BRCA2 genes. No tumor type contributes more significantly than the others amongst Dedifferentiated Liposarcoma (20%,n=4), Well-Differentiated Liposarcoma (15%,n=3), Sarcoma NOS (10%,n=2), Undifferentiated Pleomorphic Sarcoma (10%,n=2), Myxofibrosarcoma (10%,n=2) and Embryonal Rhabdomyosarcoma (10%,n=2) ending with 1 (5%) sample in each of Intimal Sarcoma, Synovial Sarcoma, Spindle Cell Rhabdomyosarcoma, Pleomorphic Liposarcoma and Rhabdomyosarcoma.

Category D: 70 samples having co-occurrence of T53 missense mutations plus at least 1 to 13 additional alterations in TP53 (truncating mutations or deletions), ATRX, RB1, CDKN2A, MAP2K4, TERT, ERBB4, NF1, PIK3CA, ATM and MYC genes (amongst the more frequent). They are significantly enriched in Leiomyosarcoma (40%,n=28) and Undifferentiated Pleomorphic Sarcoma (21%,n=15) followed by Angiosarcoma (11%,n=8), Pleomorphic Liposarcoma (6%,n=4), Solitary Fibrous Tumor/Hemangiopericytoma (4%,n=3), Pleomorphic Rhabdomyosarcoma (3%,n=2), Embryonal Rhabdomyosarcoma (3%,n=2) to end with 1 (1.4%) sample in each of Radiation-Associated Sarcoma, Sarcoma NOS, Rhabdomyosarcoma, Intimal Sarcoma, Fibrosarcoma, Alveolar Soft Part Sarcoma, Alveolar Rhabdomyosarcoma and Myxofibrosarcoma.

Category E: 21 samples having exclusive T53 missense mutations. They are significantly enriched in Leiomyosarcoma (71%, n=15) followed by Undifferentiated Pleomorphic Sarcoma (10%,n=2) and 1 (5%) sample in each of Sarcoma NOS, Synovial Sarcoma, Angiosarcoma and Liposarcoma.

Category F: 32 samples with co-occurrence of CDK4 amplifications or TP53 truncation mutations plus at least 1 to 15 additional alterations in ATRX, RB1, CDKN2A, TP53, MAP2K4, TERT, ERBB4, NF1, ATM and MYC (amongst the more frequent). They are significantly enriched in Leiomyosarcoma (41%, n=13) and Undifferentiated Pleomorphic Sarcoma (28%,n=9) followed by Angiosarcoma (13%,n=4) followed by Pleomorphic Liposarcoma (6%,n=2) and 1 (3%) sample for each of Embryonal Rhabdomyosarcoma, Spindle Cell Rhabdomyosarcoma, Sarcoma NOS and Myxofibrosarcoma.

Category G: 9 samples with only TP53 truncating mutations not significantly enriched in Leiomyosarcoma (56%,n=5) and 1 (11%) sample in each of Solitary Fibrous Tumor/Hemangiopericytoma, Rhabdomyosarcoma, Fibrosarcoma and Pleomorphic Liposarcoma.

Category H: 53 samples with co-occurrence of at most 6 alterations within genes such as ATRX, RB1, CDKN2A, TP53, MAP2K4, TERT, ERBB4, NF1, PIK3CA, ATM, KMT2D and ARID1A. They are significantly

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enriched in Undifferentiated Pleomorphic Sarcoma (34%,n=18) and Leiomyosarcoma (34%,n=18) followed by Myxofibrosarcoma (8%,n=4), Perivascular Epithelioid Cell Tumor (6%,n=3), Sarcoma NOS (4%,n=2) and 1 (2%) sample for each of Sclerosing Epithelioid Fibrosarcoma, Synovial Sarcoma, Embryonal Rhabdomyosarcoma, Solitary Fibrous Tumor/Hemangiopericytoma, Angiosarcoma, Intimal Sarcoma, Rhabdomyosarcoma and Pleomorphic Liposarcoma.

Category I: 157 samples with co-occurrences of at most 14 alterations within 99 genes of which the most frequent are MAP2K4, ERBB4, NF1, PIK3CA, ATM, MYC, WT1, KMT2D, ARID1A, NOTCH1, NOTCH2, PTEN, ALK, EP300, FLT3, FLT4, KMT2D, ROS1, SMARCA4, TET2, TSC2. They are significantly enriched in Angiosarcoma (13%,n=20), Leiomyosarcoma (11%,n=17), Desmoplastic Small-Round-Cell Tumor (10%,n=16), Undifferentiated Pleomorphic Sarcoma (10%,n=16), Solitary Fibrous Tumor/Hemangiopericytoma (10%,n=15), Synovial Sarcoma (8%,n=13) and Myxoid/Round-Cell Liposarcoma (4%,n=11) followed by Embryonal Rhabdomyosarcoma (4%,n=6), Sarcoma, NOS (4%,n=6), Rhabdomyosarcoma (3%,n=4), Clear Cell Sarcoma(3%,n=4), Epithelioid Hemangioendothelioma (2%,n=3), Radiation-Associated Sarcoma (2%,n=3), Myxofibrosarcoma (2%,n=3), Alveolar Rhabdomyosarcoma (2%,n=3), 2 (1%) samples for each of Well-Differentiated Liposarcoma, Round Cell Sarcoma NOS, Fibrosarcoma, Epithelioid Sarcoma, Proximal-Type Epithelioid Sarcoma and Low-Grade Fibromyxoid Sarcoma, 1 (<1%) sample for each of Liposarcoma, Pleomorphic Rhabdomyosarcoma, Perivascular Epithelioid Cell Tumor, Alveolar Soft Part Sarcoma and Dedifferentiated Liposarcoma.

Category A, C, D, F, H and I are denoted as “Many Drivers” while Categories B, E and G are “Mono / Few Drivers”. We have summarized the percentage of cluster enrichment of each tumor for the most frequent Sarcoma histotypes in the table below:

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Supplementary Figure 6: Proportion of samples with and without alterations according to the NGS sample size

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Supplementary Figure 7: Objective responses to targeted therapies in STS

Supplementary Figure -6. Objective Responses to targeted therapies in advanced soft-tissue sarcomas. (A-B) Response to an investigational BRAF inhibitor in a 42-year old female with BRAF- mutated undifferentiated pleomorphic sarcomas after 2 cycles of treatment (B). (C-D) Response to an investigational mTOR inhibitor in a 75-year old female with IGF1R-amplified leiomyosarcoma after 2 cycles of treatment (D)

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Supplementary Figure 7: Design of the MULTISARC study

Arm/ Target Drus(s)

EGFR/HER2 Dual EGFR/HER2 inhibitor

MET amp/mutation MET inhibitor

ALK/ROS1 amp/rearrangement ALK/ROS1 inhibitor

BRCA1 mutation PARP inhibitor

FGFR1/2/3, FRS2 mutation/ampl FGFR inhibitor

RICTOR amplification, TSC1/2 loss, mTORC1/mTORC2 inh PTEN loss

CDK4,CCND1/2/3 amplification CDK4/6 inhibitor

SMO/PTCH1 Smo inhibitor

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NF1 loss MEK inhibitor

cKIT, CSF1R mutation/amplification KIT inhibitor

TP53 mutation/loss, CDKN2A, RB1 loss Wee inhibitor

NOTCH1/2/3 Gamma-secretase inhibitor

NTRK1/2/3 NTRK inhibitor

High mutational load PDL1 + PARP inh

KDR mutation VEGFR inhibitor

AKT mutation AKT inhibitor

PI3KCA mutation PI3KCA inhbitor

Supplementary Figure 7. Design of the MULTISARC study. SCREENING – TUMOR QUALIFICATION:Upon signature of consent, eligible patient will be registered on study centrally following study procedures. For each patient: Frozen tumor material and blood samples will be obtained for genetic profiling. INCLUSION – RANDOMIZATION: FIRST LINE TREATMENT: In case of material qualification, at Cycle 1 Day 1 of 1st line anthracycline regimen, eligible patients will be centrally included and randomized on study following study procedures. Both first line clinical management and bioinformatics analysis will be performed blinded to randomization results. After tumor assessment at the end of first line chemotherapy (for a maximum of 6 cycles) and regardless of tumor response as per RECIST v1.1, investigator sites will be informed of randomization results: (i) Patients randomized in Arm B (treatment strategy not based on the use of NGS) will be treated at the discretion of the investigator and followed up until death or study termination whichever occurs first. Note that for these patients, none subsequent NGS-guided therapy will be allowed during follow-up unless the patient has exhausted all available therapies and sponsor agree to share NGS data. (ii) Patients randomized in Arm A (treatment strategy based on the use of NGS results) will be discussed within a multidisciplinary tumor board (molecular tumor board-MTB) which aims at discussing the genomic profiles and at providing a therapeutic decision for each patient. This MTB involves clinical oncologists, pathologists, biologists, and bioinformaticians. Patients for whom no targetable genomic alteration (ie. genomic alteration that cannot be targeted by one of the drug available in the present study) has been identified will be treated at the discretion of the investigator and followed up until death or study termination whichever occurs first. Note: for these patients, genetic profiles could be discussed at MTB whenever a change in therapy is required. Patients for whom a targetable genomic alteration (ie. genomic alteration that can be targeted by one of the drug available in the present study) has been identified will be proposed to enter in one of the subsequent phase II single-arm sub-trial. These patients will have to sign a second specific informed consent form in order to initiate all the selection and baseline assessments before targeted treatment initiation.

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