CCOMPOMPAASSS®S®Clinical HisClinical Histtoryory78-year-old female with a history of unexplained pancytopenia, including transfusion-dependent anemia. A bone marrow biopsy done 2 years prior atan outside institution was reportedly normal. Accompanying CBC report shows WBC 4.3 K/uL, Hgb 10.1 g/dL, MCV 94.7 fL, RDW 15.8%, platelets 129K/uL with a differential count of neutrophils 29.4, lymphocytes 62.7%, monocytes 7.9%. Evaluate for myelodysplastic syndrome.

FINAL DIAFINAL DIAGNOGNOSIS:SIS:NORMOCELLNORMOCELLULAR BONE MARROULAR BONE MARROW WITH MAW WITH MATURING TRILINEATURING TRILINEAGE HEMAGE HEMATTOPOIESIS AND MOLECULAR EVIDENCE OF CLOPOIESIS AND MOLECULAR EVIDENCE OF CLONAL HEMAONAL HEMATTOPOIESISOPOIESIS

CCompromprehensivehensive Ase AssessessmentsmentBone marrow morphology shows no evidence of an overt hematopoietic malignancy. There is no significantdysplasia. Given the patient's concerning history of unexplained pancytopenia with transfusion-dependentanemia, however, genetic studies are performed for further evaluation. While cytogenetic studies arenormal, next-generation sequencing is positive for pathogenic and/or likely pathogenic mutations in theASXL1, TET2, and ZRSR2 genes, consistent with clonal hematopoiesis. This patient may be best thought ofas having a clonal cytopenia of undetermined significance (CCUS), a term that has been proposed forcytopenic patients who do not currently meet diagnostic criteria for myelodysplastic syndrome (MDS), butnevertheless harbor a somatic mutation in a MDS-associated gene (Kwok B, et al. Blood2015;126:2355-2361). While the natural history of CCUS is still largely undefined, a recent study showed thatpatients with CCUS have a significantly higher probability of developing MDS than patients withoutevidence of clonality (Malcovati L, et al. Abstract. Blood 2016;128:298). Accordingly, the NationalComprehensive Cancer Network (NCCN) Guidelines for MDS recommend clinical follow-up every 6 monthsfor patients with CCUS.

The work-up of this case included review of all submitted clinical and laboratory information, which inconjunction with the morphologic findings, helped direct the selection of laboratory testing.

MorphologyMorphologyBone marrow aspirate smears, touch preparations, clot section, and core biopsy:- Normocellular bone marrow with maturing trilineage hematopoiesis, normal iron stores, a non-diagnosticlymphoid aggregate, and no increase in blasts

Peripheral blood smears:- Pancytopenia

FloFlow Cw CytytometryometryNo immunophenotypic evidence of a monoclonal B-cell, aberrant T-cell, or increased blast population

CCytytogenetics/FISHogenetics/FISHChromosome analysis showed a normal female karyotype in 20 of 20 cells examined. Fluorescence in situhybridization utilizing probes specific for abnormalities commonly associated with MDS (including -5/5q-,-7/7q-, +8, and 20q-) showed no abnormalities in 200 nuclei per probe examined.

Myeloid Molecular ProfilePathogenic alterations is DETECTED in the ASXL1 and ZRSR2 genes. Likely pathogenic alteration isDETECTED in the TET2 gene.

Electronically Signed ByBrian KBrian Kwwok, M.Dok, M.D..Director of Hematopathology

Date

For detailed information on any of the tests performed, please refer to the individual test reports.

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Myeloid Molecular Profile

CLINICAL DATA 78-year-old female with a history of unexplained pancytopenia, including transfusion-dependent anemia. A bone marrowbiopsy done 2 years prior at an outside institution was reportedly normal. Accompanying CBC report shows WBC 4.3 K/uL,Hgb 10.1 g/dL, MCV 94.7 fL, RDW 15.8%, platelets 129 K/uL with a differential count of neutrophils 29.4, lymphocytes 62.7%,monocytes 7.9%. Evaluate for myelodysplastic syndrome.

NGS RESULNGS RESULTTSSPathogenic alterations are DETECTED in the ASXL1 and ZRSR2 genes. Genomic alteration of uncertain significance isDETECTED in the TET2 gene.

GeneGene ExExons Tons Tesestteded Genomic AltGenomic Altereraation(tion(ss)) MutaMutation Efftion Effectect Allele FrAllele Frequencequencyy PPaathogenicthogenic

ASXL1 12-13 c.2637_2641delTACTA; p.D879Efs*13 FRAMESHIFT 38% YES

TET2 ALL c.3881A>G; p.Y1294C MISSENSE 41% UNCERTAIN

ZRSR2 2-5,7-11 c.73G>T; p.E25* NONSENSE 79% YES

Genes AnalyGenes Analyzzed with no Genomic Alted with no Genomic Altereraations Dettions Detectected bed by NGS:y NGS:

BCOR BRAF CALR CBL CEBPA CSF3R DDX41 DNMT3A ETNK1 ETV6

EZH2 GATA2 GNAS GNB1 IDH1 IDH2 JAK2 KIT KRAS MPL

NF1 NPM1 NRAS PDGFRA PHF6 PPM1D PTPN11 RAD21 RUNX1 SETBP1

SF3B1 SH2B3 SMC1A SMC3 SRSF2 STAG2 STAT3 STAT5B TP53 U2AF1

WT1

NGS IntNGS Interprerpretaetation:tion:A genomic alteration in the ASXL1 gene is detected (c.2637_2641delTACTA; p.D879Efs*13). This frameshift alteration has notbeen previously reported to our knowledge, but is expected to be pathogenic.

A genomic alteration in the TET2 gene is detected (c.3881A>G; p.Y1294C). This missense alteration has not been previouslyreported to our knowledge, and the clinical significance is uncertain

A genomic alteration in the ZRSR2 gene is detected (c.73G>T; p.E25*). This nonsense alteration has not been previouslyreported to our knowledge, but is expected to be pathogenic.

Clinical Summary of MutaClinical Summary of Mutatted Genesed Genes

ASXL1 Alterations

ASXL1, located on 20q11, encodes for a member of the polycomb family of chromatin-binding proteins, which are involved inepigenetic regulation of gene expression. ASXL1 mutations are found in 37% of chronic myelomonocytic leukemia (PMID:27385790), 32% of blastic plasmacytoid dendritic cell neoplasm (PMID: 24072100), 29% of refractory anemia with ringsideroblasts associated with marked thrombocytosis (PMID: 26874914), 23% of myelodysplastic syndrome (PMID: 24220272),22% of primary myelofibrosis (PMID: 23619563), 17% of systemic mastocytosis (PMID: 27214377), 12% of polycythemia vera,11% of essential thrombocythemia (Tefferi A, et al. Blood Advances 2016;1:21-30), and 11% of acute myeloid leukemia (PMID:27288520).

ASXL1 mutations are associated with an unfavorable prognosis in myelodysplastic syndrome, independent of IPSS, IPSS-R,

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Myeloid Molecular Profile

age, and other gene mutations. When mutation status is integrated into the survival analysis by IPSS risk categories, ASXL1mutations are reported to shift the survival curve of the IPSS risk category to resemble that of the next highest IPSS risk level(PMID: 21714648). Accordingly, the NCCN Guidelines state that gene mutation analysis can refine the prognosis ofmyelodysplastic syndrome in patients risk stratified by the IPSS or IPSS-R, and may be helpful in patients predicted to haveintermediate risk (NCCN Guidelines for Myelodysplastic Syndromes). ASXL1 mutations are independently associated withunfavorable outcomes and shorter survival after hematopoietic stem cell transplantation in patients with myelodysplasticsyndrome and acute myeloid leukemia evolving from myelodysplastic syndrome (PMID: 27601546). ASXL1 mutations are alsoassociated with an unfavorable prognosis in chronic myelomonocytic leukemia (only nonsense and frameshift mutations)(PMID: 27385790), refractory anemia with ring sideroblasts associated with marked thrombocytosis (PMID: 26874914),primary myelofibrosis (PMID: 23619563), polycythemia vera (Tefferi A, et al. Blood Advances 2016;1:21-30), systemicmastocytosis (PMID: 27214377), and acute myeloid leukemia with wild-type FLT3-ITD and normal karyotype or intermediate-risk cytogenetic abnormalities (PMID: 22417203). ASXL1 mutations are reported to be highly specific for secondary acutemyeloid leukemia, and may also be helpful in identifying a subset of elderly patients with de novo acute myeloid leukemiawith worse clinical outcomes (PMID: 25550361).

Somatic mutations in leukemia-associated genes, particularly ASXL1, DNMT3A, or TET2, can also be found in about 10% ofelderly individuals without known hematologic malignancy, a condition known as clonal hematopoiesis of indeterminatepotential (CHIP). Most of these individuals, however, lack cytopenias and have only a single mutation with low allelefrequency. The presence of a somatic mutation is associated with increases in the risk of developing a hematologicmalignancy and in all-cause mortality, with the risk of developing a hematologic malignancy increased by a factor of nearly50 in individuals with allele frequency of 10% or more (PMID: 25426837). Somatic mutations in leukemia-associated genesare also commonly found in patients with unexplained cytopenias who do not meet diagnostic criteria for myelodysplasticsyndrome, a condition known as clonal cytopenias of undetermined significance (CCUS) (PMID: 26429975 and PMID:26392596).

TET2 Alterations

TET2, located on 4q24, encodes for a protein that plays a critical role in DNA methylation by converting methylcytosine to5-hydroxymethylcytosine. TET2 mutations are found in 44% of chronic myelomonocytic leukemia (PMID: 27385790), 36% ofblastic plasmacytoid dendritic cell neoplasm (PMID: 24072100), 33% of myelodysplastic syndrome (PMID: 24220272), 29% ofsystemic mastocytosis (PMID: 27214377), 22% of polycythemia vera, 16% of essential thrombocythemia (Tefferi A, et al. BloodAdvances 2016;1:21-30), 15% of acute myeloid leukemia (PMID: 27288520), 10% of primary myelofibrosis (PMID: 23619563),and 10% of refractory anemia with ring sideroblasts associated with marked thrombocytosis (PMID: 26874914).

TET2 mutations are associated with an increased response to hypomethylating agents when the allele frequency is >10% andASXL1 is not mutated (PMID: 25224413). TET2 mutations are also associated with unfavorable outcomes and shorter survivalafter hematopoietic stem cell transplantation in patients with myelodysplastic syndrome (PMID: 25092778). Co-mutation ofTET2 and SRSF2 is reported to be highly predictive of myelodysplasia with monocytosis, such as chronic myelomonocyticleukemia (PMID: 24970933). In acute myeloid leukemia with wild-type FLT3-ITD and normal karyotype or intermediate-riskcytogenetic abnormalities, TET2 mutations are associated with an unfavorable prognosis (PMID: 22417203).

Somatic mutations in leukemia-associated genes, particularly ASXL1, DNMT3A, or TET2, can also be found in about 10% ofelderly individuals without known hematologic malignancy, a condition known as clonal hematopoiesis of indeterminatepotential (CHIP). Most of these individuals, however, lack cytopenias and have only a single mutation with low allelefrequency. The presence of a somatic mutation is associated with increases in the risk of developing a hematologicmalignancy and in all-cause mortality, with the risk of developing a hematologic malignancy increased by a factor of nearly50 in individuals with allele frequency of 10% or more (PMID: 25426837). Somatic mutations in leukemia-associated genesare also commonly found in patients with unexplained cytopenias who do not meet diagnostic criteria for myelodysplasticsyndrome, a condition known as clonal cytopenias of undetermined significance (CCUS) (PMID: 26429975 and PMID:26392596).

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Myeloid Molecular Profile

ZRSR2 Alterations

ZRSR2, located on Xp22.1, encodes for a protein that associates with the U2 auxiliary factor heterodimer, which is involving insplicing. ZRSR2 mutations are found in 8% of myelodysplastic syndrome (PMID: 24220272), 8% of blastic plasmacytoiddendritic cell neoplasm (PMID: 24072100), 4% of chronic myelomonocytic leukemia (PMID: 27385790), 2% ofmyeloproliferative neoplasm (PMID: 21909114), and 1% of acute myeloid leukemia (PMID: 27288520).

ZRSR2 mutations are associated with an unfavorable prognosis in myelodysplastic syndrome (NCCN Guidelines forMyelodysplastic Syndromes). ZRSR2 mutations are also reported to be highly specific for secondary acute myeloid leukemia,and may also be helpful in identifying a subset of elderly patients with de novo acute myeloid leukemia with worse clinicaloutcomes (PMID: 25550361).

Electronically Signed ByHans DecherHans Decher,,Sr Mgr Marketing Operations

Date

This test was developed by and its performance characteristics determined by the performing laboratory. All tests are performed at Genoptix, Inc., unless otherwise specified in the report. The test has not been cleared or approved by the US Food andDrug Administration. The FDA has determined that such clearance or approval is not necessary. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical LaboratoryImprovement Amendments of 1988 (CLIA-88) as qualified to perform high complexity clinical testing.

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Clinical Utility:Clinical Utility:

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by cytopenias,ineffective hematopoiesis, morphologic dysplasia, and a variable risk of transformation to acute myeloid leukemia (AML).Establishing a diagnosis of MDS in a cytopenic patient is often challenging as quantification of dysplasia and blasts can besubjective and prone to wide interobserver variation even among expert hematopathologists.1 Targeted sequencing canidentify 1 or more somatic mutation in 80-90% of MDS patients, and the National Comprehensive Cancer Network (NCCN)Guidelines recommends molecular testing of bone marrow or peripheral blood for MDS-associated gene mutations inappropriate clinical contexts as an aid in diagnosis and risk stratification.2-4

• Mutations in TP53, EZH1, ETV6, RUNX1, and ASXL1 are predictors of poor overall survival in patients with MDS,independently of established risk factors.5

• Mutations in BCOR, DNMT3A, IDH1, NRAS, splicing factor genes (SRSF2, U2AF1, ZRSR2), and cohesion complex genes(RAD21, SMC1A, SMC3, STAG2); as well as a high total number of mutations; have also been shown to be associated withan unfavorable prognosis in MDS. SF3B1 mutations are highly predictive for the presence of ring sideroblasts, and areassociated with a favorable prognosis.2-9

• TET2 mutations are associated with an increased response to hypomethylating agents in patients with MDS when theallele frequency is >10% and ASXL1 is not mutated.10

• TP53 mutations are associated with an unfavorable prognosis and decreased response to lenalidomide in patients withMDS with isolated del(5q), and TP53 mutation evaluation is recommended in the 2016 revision of World HealthOrganization (WHO) classification to help identify an adverse prognostic subgroup in this generally favorable prognosisMDS entity.11,12

• Mutations in TP53, TET2, or DNMT3A are predictive of poor outcomes in MDS after hematopoietic stem celltransplantation.13

• MDS-associated gene mutations are also commonly found in patients with potential pre-phase of MDS, including clonalcytopenias of undetermined significance and preclinical MDS syndrome.14,15

Molecular profiling can also help in the diagnostic evaluation and risk stratification of other myeloid neoplasms.

• Mutations in key driver genes are included as diagnostic criteria for myeloproliferative neoplasms (MPN) in the 2016revision of the WHO classification: JAK2 V617F or JAK2 exon 12 for polycythemia vera; JAK2, CALR, or MPL for primarymyelofibrosis (PMF) and essential thrombocythemia; and CSF3R for chronic neutrophilia leukemia.12

• Mutations in ASXL1, EZH2, SRSF3, and IDH1/2 are associated with an unfavorable prognosis in PMF.16

• Targeted sequencing can identify 1 or more somatic mutation in 90% of chronic myelomonocytic leukemia (CMML)patients, and the presence of mutations is included as one of the diagnostic criteria for CMML in the 2016 revision of theWHO classification. Mutations in RUNX1, NRAS, SETBP1, and ASXL1 (nonsense and frameshift) are associated with anunfavorable prognosis.12,17

• Mutations in SETBP1 and/or ETNK1 are found in up to a third of patients with atypical chronic myeloid leukemia, a rareand difficult to diagnosis MDS/MPN subtype.12

• Myeloid neoplasms that occur on the background of a predisposing germline CEBPA, DDX41, RUNX1, ETV6, or GATA2mutation are included as entities in the 2016 revision of the WHO classification.12

• Mutations in key driver genes define distinct genomic subgroups of AML. These include the currently defined AMLsubgroups in the WHO classification as well as 3 other genomic subgroups: AML with mutations in genes encodingchromatin, RNA splicing regulators, or both; AML with TP53 mutations, chromosomal aneuoploidies, or both; and AMLwith IDH2R172 mutations. Chromatin-spliceosome and TP53-aneuploidy AML are associated with poor outcomes.12,18

• Mutations in SFSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 are reported to be highly specific for secondaryAML, and may also be helpful in identifying a subset of elderly patients with de novo AML with worse clinical outcomes.19

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RRefefererencences:es:1. Font P, et al. Inter-observer variance with the diagnosis of myelodysplastic syndromes (MDS) following the 2008 WHO classification. Ann Hematol 2013;92:19-24.2. National Comprehensive Cancer Network (NCCN) Practice Guidelines in Oncology, Myelodysplastic Syndromes. Version 2.2017.3. Papaemmanuil E, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood 2013;122:3616-27.4. Haferlach T, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia 2014;28:241-247.5. Bejar R, et al. Clinical effects of point mutations in myelodysplastic syndromes. N Engl J Med 2011;364:2496-506.6. Damm F, et al. BCOR and BCORL1 mutations in myelodysplastic syndromes and related disorders. Blood 2013;122:3169-3177.7. Patnaik MM, et al. Differential prognostic effect of IDH1 versus IDH2 mutations in myelodysplastic syndromes: a Mayo Clinic study of 277 patients. Leukemia 2012;26:101-105.8. Thota S, et al. Genetic alterations of the cohesin complex genes in myeloid malignancies. Blood 2014;124:1790-1798.9. Malcovati L, et al. SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts. Blood 2015;126:233-241.

10. Bejar R, et al. TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients. Blood 2014;124:2705-2712.11. Jadersten M, et al. TP53 mutations in low-risk myelodysplastic syndromes with del(5q) predict disease progression. J Clin Oncol 2011;29:1971-1979.12. Arber DA, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016; 127:2391-405.13. Bejar R, et al. Somatic mutations predict poor outcome in patients with myelodysplastic syndrome after hematopoietic stem-cell transplantation. J Clin Oncol 2014;32:2691-2698.14. Kwok B, et al. MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance. Blood 2015;126:2355-2361.15. Cargo C, et al. Targeted sequencing identifies patients with preclinical MDS at high risk of disease progression. Blood 2015;126:2362-2365.16. Vannucchi AM, et al. Mutations and prognosis in primary myelofibrosis. Leukemia 2013;27:1861-1869.17. Elena C, et al. Integrating clinical features and genetic lesions in the risk assessment of patients with chronic myelomonocytic leukemia. Blood 2016;doi:10.1182/blood-2016-05-714030.18. Papaemmanuil E, et al. Genomic classification and prognosis in acute myeloid leukemia. N Engl J Med 2016;374:2209-2221.19. Lindsley RC, et al. Acute myeloid leukemia ontogeny is defined by distinct somatic mutations. Blood 2015;125:1367-1376.

AsAssasay Summary:y Summary:This assay utilizes DNA sequencing to interrogate 44 genes known to be recurrently mutated in myeloid malignancies or areknown to occur in malignancies that may mimic the clinical presentation of some myeloid malignancies. Depending on thegene, the assay interrogates either select coding regions or entire coding regions for base substitutions, insertion/deletions,and copy number variations (CNV).

GeneGene ExExons Tons Tesestteded CNVCNV

ASXL1 12-13 No

BCOR ALL No

BRAF 1,3,6,8,10-11,13,15-18 Yes

CALR ALL Yes

CBL 8-11,13 Yes

CEBPA ALL Yes

CSF3R ALL No

DDX41 ALL No

DNMT3A 8-23 No

ETNK1 3 No

ETV6 ALL Yes

EZH2 ALL Yes

GATA2 ALL No

GNAS 8-9 No

GNB1 ALL No

IDH1 4,6 No

IDH2 4 No

JAK2 3,7,11-14,16,20 Yes

KIT 2-3,8-11,13,17-18 Yes

KRAS ALL Yes

MPL 10-11 No

NF1 ALL Yes

GeneGene ExExons Tons Tesestteded CNVCNV

NPM1 10-12 No

NRAS ALL Yes

PDGFRA 4,10-23 Yes

PHF6 ALL No

PPM1D ALL Yes

PTPN11 3,13 No

RAD21 ALL No

RUNX1 3-8 No

SETBP1 4 Yes

SF3B1 7,13-16,18,23-24 Yes

SH2B3 2 No

SMC1A ALL No

SMC3 ALL No

SRSF2 1 No

STAG2 ALL No

STAT3 20-21 No

STAT5B 16 No

TET2 ALL No

TP53 ALL Yes

U2AF1 2,6 No

WT1 ALL No

ZRSR2 2-5,7-11 No

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Methods:Methods:Patient genomic DNA, isolated from peripheral blood, bone marrow or formalin fixed paraffin embedded (FFPE) tissue, isutilized to identify relevant single nucleotide variants (SNV), insertion/deletions (indel) and copy number variations (CNV) ina panel of up to 237 reportable genes (see Profile Assay Summary). Targeted genomic regions are prepared and sequencedby Next Generation Sequencing. The genomic alterations within each of these genes are detected through proprietarybioinformatic analysis software and interpreted in conjunction with reference databases such as COSMIC and dbSNP. Qualitycontrol metrics include a minimum input of 20 ng, with an optimal input of 100ng of genomic DNA, and average meansequencing depth of 500x coverage. The limits of detection (LOD) are 5% for SNV, 10% for indels, ≥6 copies for geneamplifications, and ≤0.3 copies for homozygous gene deletions. Benign sequence variants are not reported.

LimitaLimitations:tions:• Assay failures may result from patient samples that render inadequate quantity and/or poor quality of DNA, and

specimens that do not generate sufficient sequencing coverage to facilitate accurate variant assessment.• Sequencing quality may be diminished in specific genomic regions, and a pathogenic alteration may be present and not

reported if those regions do not meet quality control criteria. These genomic alterations, if present, are available uponrequest.

Appendix:Appendix:Genoptix may include relevant clinical annotations specific to potential diagnostic, prognostic, and predictive implications asmedically appropriate for certain genes. The data supporting these clinical annotations are derived from published peer-reviewed studies, medical and scientific abstracts, guidelines, and other publicly available information. The reference sourcesare provided within the clinical annotation.

Re-assessment of genomic alteration classifications is continuous based on emerging evidence. When a change in apreviously-reported classification is warranted, appropriate communication may be generated by Genoptix.

MyMyeloid Molecular Preloid Molecular Profileofile

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