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 genes 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 - Human Genome Variation Society protein 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 GENE 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