Patient Study ID: DFCI Analysis ID: DFCI-XXX Report Date: MM/DD/YYYY MMRF_XXXXXXXX

MMRF CURECLOUD cfDNA MYELOMA PANEL

Patient Study ID: MMRF_XXXXXXXX Sample ID: XXXXXXXX_P DFCI Analysis ID: DFCI-XXX Sample Type: cfDNA from blood Other ID: N/A Collection Date: N/A Patient DOB: N/A CRSP Received Date: N/A Sex: N/A Report Electronically Signed by: XXXXX XXXXXXX, M.D. Ordering Physician: N/A Date Report Signed: MM/DD/YYYY Ordering Site: N/A Time Report Signed: HR:MM

Results interpretation performed by the Dana-Farber Cancer Institute Interpretive Genomics Program (Co-Directors Dr. Annette S. Kim and Dr. Keith L. Ligon), Department of Oncologic Pathology, 450 Brookline Ave., Boston, MA 02215 Tel:(617) 582-8764 or (617) 632- 5482. Email: [email protected]

ABOUT THE ANALYSIS

Sequencing of circulating cell free DNA (cfDNA) from blood plasma from this individual was performed and the data was analyzed to identify variants in 70 that have been previously implicated in Multiple Myeloma. This test is designed for somatic analysis of variants, including single nucleotide variants and small insertions/deletions, but it cannot adequately distinguish between somatic and germline variants and therefore some germline variants may be included. However, most germline variants will be excluded from detection and therefore germline testing should be ordered separately, if indicated. This assay is not designed to identify copy number alterations, large insertions/deletions, or structural variants such as translocations. Given very low levels of tumor DNA compared to germline DNA and other possible interfering sources, this assay has limited sensitivity for difficult to detect variants and cannot always distinguish tumor related variants from other sources of variation. Clinical correlation and orthogonal validation is recommended where possible. SAMPLE REPORT

Page 1 of 6 Patient Study ID: DFCI Analysis ID: DFCI-XXX Report Date: MM/DD/YYYY MMRF_XXXXXXXX SUMMARY OF PATHOGENIC/LIKELY PATHOGENIC VARIANTS

Gene AA Mutation Consequence HGVSp HGVSc HGVSg VAF % Total Reads DIS3 p.L767F missense_variant NP_055768.3:p.Leu767Phe NM_014953.5:c.2301A>T chr13:g.72761964T>A 3.74 2597 Hotspot mutation Pathogenic PRUNE2 splice_donor_variant . NM_001308049.1:c.983+2T>G chr9:g.76619338A>C 3.95 2960 Pathogenic DNMT3A p.L395R missense_variant NP_001307822.1:p.Leu395Arg NM_001320893.1:c.1184T>G chr2:g.25244567A>C 13.98 1774 Pathogenic BRAF p.V600E missense_variant NP_001341538.1:p.Val600Glu NM_001354609.2:c.1799T>A chr7:g.140753336A>T 2.78 3529 Hotspot mutation Theranostic FUBP1 p.Q267* stop_gained&splice_r NP_001290362.1:p.Gln267Ter NM_001303433.1:c.799C>T chr1:g.77964747G>A 2.91 3060 egion_variant Pathogenic PPM1D p.E540* stop_gained NP_003611.1:p.Glu540Ter NM_003620.4:c.1618G>T chr17:g.60663352G>T 0.9 3315 Pathogenic DNMT3A p.R882C missense_variant NP_072046.2:p.Arg882Cys NM_022552.4:c.2644C>T chr2:g.25234374G>A 10.96 2718 Hotspot mutation Pathogenic EP300 p.E1026* stop_gained NP_001420.2:p.Glu1026Ter NM_001429.4:c.3076G>T chr22:g.41152284G>T 1.94 3242 Pathogenic

Abbreviations: AA - amino acid, HGVSp - Variation Society nomenclature, HGVSc - HGVS complementary DNA nomenclature, HGVSg - HGVS genomic location nomenclature, VAF - variant allele fraction The PPM1D variant is identified at a variant allele fraction lower than the validated threshold for calling a new variant. However, it is listed here to enable future comparison.

The DNMT3A and PPM1D variants are likely derived from the myeloid compartment of the hematopoietic cells and not from the plasma cells. These may indicatedSAMPLE clonal hematopoiesis or the presence of a myeloid disorder. REPORT

Page 2 of 6 Patient Study ID: DFCI Analysis ID: DFCI-XXX Report Date: MM/DD/YYYY MMRF_XXXXXXXX DESCRIPTIONS (MMRF)

DIS3 DIS3 homolog, exosome endoribonuclease and 3'-5' exoribonuclease (Tumor Suppressor Gene) 13q21.33: DIS3 encodes an exosome ribonuclease that is expressed many tissues. DIS3 mutations are apparently unique to myeloma and are associated with t(4;14) and t(14:16) cytogenetics. (PMID: 29884741; 29789651; 30967618 ) PRUNE2 prune homolog 2 9q21.2: PRUNE2 encodes a member of the B cell CLL/lymphoma 2 and adenovirus E1B interacting family with roles in apoptosis, transformation and synaptic function. PRUNE2 a tumor suppressor that is infrequently mutated in myeloma. (PMID: 24434212) DNMT3A DNA methyltransferase 3 alpha (Tumor Suppressor Gene) 2p23.3: DNMT3A encodes a DNA methyltransferase responsible for de novo methylation of cytosine bases. DNMT3A mutations are recurrent in clonal hematopoiesis, MDS, MPN, MDS/MPN, AML, T-ALL, and peripheral T-cell lymphomas. Pathogenic mutations are truncating mutations and missense mutations in the methyltransferase domain (including hotspot p.R882) and the ADD domain. DNMT3A mutations can be found without overt disease and, in the context of known disease, can persist in morphologic remission. (PMID: 28003281; 29601269; 30138727) BRAF B-Raf proto-oncogene, serine/threonine kinase (Oncogene) 7q34: BRAF encodes for a serine/threonine kinase that activates the MEK/ERK signaling pathway. BRAF mutations are nearly universal in classical hairy cell leukemia. They are recurrent in Langerhans cell histiocytosis, Erdheim-Chester disease, and less commonly identified in myeloid neoplasms and plasma cell myeloma. Missense mutations in BRAF occur primarily in exons 11-15, corresponding to the c-terminal kinase domain. Activating mutations have been identified in codons 597, 601, and most commonly 600. Inactivating mutations in BRAF codons 594-596 also result in activated signal transduction. BRAF is an established driver mutation in myeloma associated with t(14:16) translocated cases. FDA-approved inhibitors of BRAF are available. (PMID: 29884741; 29789651; 31628267) FUBP1 far upstream element binding protein 1 (Tumor Suppressor Gene) 1p31.1: FUBP1 encodes a DNA-binding protein, which is involved in regulation of Myc and AKT-PI3K pathway. FUBP1 is mutated rarely in plasma cell myeloma. (PMID: 29884741 ) PPM1D protein phosphatase, Mg2+/Mn2+ dependent 1D (Oncogene) 17q23.3: PPM1D encodes a serine-threonine phosphatase which negatively regulates the cellular stress response and DNA damage response by dephosphorylating p53, ATM, CHK1, and CHK2. Somatic mutations in PPM1D are observed in clonal hematopoiesis and myeloid malignancies, particularly in the setting of prior chemotherapy exposure. Pathogenic mutations are truncating mutations in exon 6 resulting in loss of the degradation domain, leading to enhanced stability and activity. PPM1D mutations are enriched in patients with therapy-relatedSAMPLE myeloid neoplasms. (PMID: 29386642; 29954749; 30388424) EP300 E1A binding protein p300 (Tumor Suppressor Gene) 22q13.2: EP300 (p300; E1A-binding protein) encodes a transcriptional co-activator with histone acetyltransferase (HAT) activity. EP300 mutations are recurrent in non-Hodgkin lymphomas (e.g., diffuse large B-cell lymphoma, marginal zone lymphoma, follicular lymphoma), myeloma, and rarely recurrent in myeloid neoplasms. Pathogenic mutations are truncating and missense mutations in the HAT domain leading to loss of function. In myeloma, mutations in EP300 are associated with t(14;16). (PMID: 27465822; 29460469; 15706485; 21390126; 29713087) REPORT

Page 3 of 6 Patient Study ID: MMRF_XXXXXXXX DFCI Analysis ID: DFCI-XXX Report Date: MM/DD/YYYY SOMATIC VARIANTS OF UNCERTAIN SIGNIFICANCE (VUS) & BENIGN

Gene AA Mutation Consequence HGVSp HGVSc HGVSg VAF % Total Reads ATR intron_variant . NM_001184.4:c.3581+11G>A chr3:g.142540893C>T 0.66 1672 Uncertain significance FGFR3 p.T242M missense_variant NP_000133.1:p.Thr242Met NM_000142.4:c.725C>T chr4:g.1801729C>T 42.96 852 ASXL3 p.T329I missense_variant NP_085135.1:p.Thr329Ile NM_030632.3:c.986C>T chr18:g.33734319C>T 0.89 3157 Uncertain significance SETD2 splice_region_varian . NM_001349370.2:c.5266-8T>G chr3:g.47084390A>C 44.96 1021 t&intron_variant PRUNE2 5_prime_UTR_variant . NM_001308049.1:c.-2G>C chr9:g.76687652C>G 0.55 3461 Uncertain significance

Abbreviations: AA - amino acid, HGVSp - Human Genome Variation Society protein nomenclature, HGVSc - HGVS complementary DNA nomenclature, HGVSg - HGVS genomic location nomenclature, VAF - variant allele fraction The ASXL3, ATR, and PRUNE2 variants are identified at variant allele fractions lower than the validated threshold for calling a new variant. However, they are listed here to enable future comparison.

Of note, the listed variant in FGFR3 has been noted at a very low frequency in the general population in SNP databases.

CLINICAL TRIALS

BRAF NP_001341538.1:p.Val600Glu :

NCT03091257 : Phase 1; A Study of Dabrafenib (BRAF inhibitor) and/or Trametinib (MEK inhibitor) in Patients With Relapsed and/or Refractory Multiple Myeloma..

NCT02465060 : Phase 2; NCI-MATCH: Targeted Therapy Directed by Genetic Testingin Treating Patients With Advanced Refractory Solid Tumors or Lymphomas. Subprotocol R (BRAF fusion or BRAF non-V600 mutation), will receive MEK inhibitor Trametinib..

NCT02693535 : Phase 2; TAPUR: Testing the Use of Food and Drug Administration (FDA) Approved Drugs That Target a Specific Abnormality in a TumorSAMPLE Gene in People With Advanced Stage Cancer (TAPUR), Group 13- Participants with BRAF mutations will receive regorafenib. Regorafenib is an oral multi-kinase inhibitor..

NCT03732703 : MyDRUG-Sub-Protocol C1: Patients with the presence of RAF/RAS mutation receive Cobimetinib in combination with ixazomib, pomalidomide and dexamethasone (IPd).

For dynamic information on eligibility for clinical trials on the basis of mutational profiling, please refer to https://www.mycancergenome.org/ and https://clinicaltrials.gov/REPORT.

Page 4 of 6 Patient Study ID: DFCI Analysis ID: DFCI-XXX Report Date: MMRF_XXXXXXXX MM/DD/YYYY QUALITY CONTROL

Sample: PASS Minimum Passing Metrics

Metric Minimum passing value Raw depth >60,000x Duplex depth >400x cfDNA input >10ng

METHODS

Technical Methods This assay was developed and is performed by the Clinical Research Sequencing Platform at the Broad Institute. The assay is generated on extracted circulating cell free DNA (cfDNA) from plasma and utilizes targeted hybrid capture methodology to profile a 300Kb Multiple Myeloma specific panel. Library preparation is performed using a commercially available kit provided by KAPA Biosystems (KAPA HyperPrep Kit with Library Amplification product KK8504) and IDT duplex UMI (unique molecular index) adapters. Unique 8-base dual index sequences embedded within the p5 and p7 primers are added during PCR. All cfDNA derived libraries are sequenced on an Illumina sequencing platform with 151 , paired-end reads to meet a goal of 80,000x raw coverage with a minimum depth of 60,000x raw coverage. An alignment pipeline aggregates all data from a particular sample into a single BAM file which will include all reads, all bases from all reads, and original/vendor-assigned quality scores from which coverage is determined. Variant detection is performed using a custom cloud based pipeline with duplex UMI error correction, GATK Mutect 2 and specialized filtering to reduce the presence of germline and false positive events. Output includes variants contained within the exonic regions of the following genes + 10 bp exon padding.

ACTG1 ATM CDKN1B EGR1 HIST1H1E KDM5C LTB NFKBIA PRDM1 SF3B1 AKT1 ATR CDKN2C EP300 HIST1H3G KDM6A MAF NOTCH1 PRKD2 SP140 ALK ATRX CREBBP ETV4 HIST1H3H KMT2A MAFB NRAS PRUNE2 TET2 ARID1A BCL7A CXCR4 FAM46C IDH1 KMT2B MAX NRM PTPN11 TGDS ASXL1 BRAF CYLD FGFR3 IDH2 KMT2C MYD88 PIK3CA RASA2 TP53 ASXL2 CCND1 DIS3 FUBP1 IGF1R KMT2D NCOR1 PPM1D RB1 TRAF3 ASXL3 CDH4SAMPLE DNMT3A HIST1H1C IRF4 KRAS NF1 PRAMEF2 SETD2 ZFHX3 Interpretation and Variant Annotation Methods DFCI interpretation is performed using the MERCURY reporting system (developed and validated by IntegraGen SA) to provide variant calls which are realigned to human reference sequence GRCh38. Ensembl’s VEP (Variant Effect Predictor, release VEP99) program processes variants from MAF file. This tool annotates variants and attempts to predict effects on relevant transcripts and . It takes into account data available in the gnomAD, the 1000 Genomes Project and the Kaviar databases. Moreover, anREPORT in-house database enables filtration of many sequencing artefacts. Five bioinformatics algorithms for pathogenicity are available to inform the functional, molecular and phenotypic consequences of coding and non-coding SNPs. This includes DANN, FATHMM, MutationTaster, SIFT and Polyphen. The clinical and pathological significance from the ClinVar database is considered. Other information, including quality score, homozygote/heterozygote status, count of variant allele reads, and presence of the variant in the COSMIC database are available to the signing pathologist. RegulomeDB is used to annotate SNPs in known and predicted regulatory elements in the intergenic regions. Limitations This test is designed for somatic analysis of variants and is not intended to provide germline variant calls. This assay applies filtering to enrich for somatic variants but cannot always distinguish between somatic and germline events. Germline testing should be ordered separately, as indicated.

Page 5 of 6 Patient Study ID: DFCI Analysis ID: DFCI-XXX Report Date: MM/DD/YYYY MMRF_XXXXXXXX

Sequence data are aligned to NCBI human reference sequence GRCh37 after discarding low quality sequences. Reads that align to more than one region of the reference genome, reads with low alignment scores, and bases with low quality scores are excluded from variant calling and variants outside of regions targeted by the capture bait set are not called or reported. With at least 20ng of cfDNA input, this assay has been validated to detect 91.0% of SNVs present at 1% simulated tumor fraction with a false discovery range of 0.14-2.94 false positives per Mb. Short insertions and deletions have limited sensitivity using this assay (<30% detection sensitivity). The assay will not detect genomic rearrangements, epigenetic changes, copy number variants or other structural genomic changes. For somatic and mosaic samples, the assay may show more limited detection of all variant types when at low allelic frequency. Limited quantity cfDNA samples are not able to undergo confirmatory fingerprint germline genotyping to rule out sample mishandling events during the course of collection or processing. As a result, there is a small possibility that undetected mishandling events could occur. We recommend confirmatory testing of events reported by this assay. Genomic alterations detected may be associated with activity of certain FDA-approved drugs; however, the agents listed in this report may have little or no evidence in the patient’s tumor type. Neither the therapeutic agents nor the trials identified are ranked in order of potential or predicted efficacy for this patient, nor are they ranked in order of level of evidence for this patient’s tumor type.

LABORATORY INFORMATION

CRSP, LLC. is authorized under the Clinical Laboratory Improvement Amendments (CLIA) to develop and perform laboratory testing. This test was developed and its performance characteristics determined by CRSP, LLC. The U.S. Food and Drug Administration (FDA) has not approved or cleared this test; however, FDA clearance or approval is not currently required for clinical use. The results are not intended to be used as the sole means for clinical diagnosis or patient management decisions. Confirmation testing of variants by alternative methods is recommended. Lab Director: Heidi Rehm PhD, FACMG 320 Charles St, Cambridge, MA 02141 [email protected] CLIA: 22D2055652 CAP: 8707596 Commonwealth of Massachusetts Clinical Laboratory License: 3306 SAMPLE REPORT

Clinical report generated using developed by IntegraGen SA. All rights reserved.

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