Patient Report FINAL. II. Tier 2 Variants (Variants of unknown significance in myeloid malignancies): NONE DETECTED

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2 of patients with essential thrombocythemia (ET), and 40-50% of patients with primary myelofibrosis (PMF) (Baxter et al., 2005; James et al., 2005; Kralovics et al., 2005; Levine et al., 2005; Pietra et al., 2008; Scott et al., 2007). This particular JAK2 mutation (p.val617phe) occurs in the pseudokinase (JH2) domain and leads to activation of the JAK-STAT pathway signaling. PMF patients with JAK2 mutations have a worse outcome compared to PMF patients with CALR mutations, but a better outcome compared to the group of so-called 'triple negative' patients who lack JAK2, CALR and MPL mutations (Rumi et al., 2014b). In ET, JAK2 mutations are associated with a higher risk of thrombosis compared to patients with CALR mutations (Klampfl et al., 2013; Rotunno et al., 2014; Rumi et al., 2014a). Another recent study of a cohort of 197 young ET patients showed JAK2 mutations significantly correlate with higher thrombotic risk, shorter overall survival and event-free survival (Palandri et al., 2015). 2. ETNK1 c.731a>g, p.asn244ser (NM_ ) Variant Frequency: 7.1% Interpretation: ETNK1 encodes an ethanolamine kinase, which catalyzes the first step of the de novo phosphatidylethanolamine (PE) biosynthesis pathway. Somatic mutations of ETNK1 are very rare in MPN. This particular missense mutation (p.asn244ser) is a recurrent ETNK1 mutation in myeloid malignancies (Gambacorti-Passerini et al., 2015; Lasho et al., 2015). One study suggests that this mutation impairs the catalytic activity of ETNK1 (Gambacorti-Passerini et al., 2015). The prognostic significance of ETNK1 mutations in MPN is unknown at this time. II. Tier 2 Variants (Variants of unknown significance in myeloid malignancies): NONE DETECTED References: 1.Baxter, E.J., L.M. Scott, P.J. Campbell, C. East, N. Fourouclas, S. Swanton, G.S. Vassiliou, A.J. Bench, E.M. Boyd, N. Curtin, M.A. Scott, W.N. Erber, A.R. Green, and P. Cancer Genome Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 365: Gambacorti-Passerini, C.B., C. Donadoni, A. Parmiani, A. Pirola, S. Redaelli, G. Signore, V. Piazza, L. Malcovati, D. Fontana, R. Spinelli, V. Magistroni, G. Gaipa, M. Peronaci, A. Morotti, C. Panuzzo, G. Saglio, E. Usala, D.W. Kim, D. Rea, K. Zervakis, N. Viniou, A. Symeonidis, H. Becker, J. Boultwood, L. Campiotti, M. Carrabba, E. Elli, G.R. Bignell, E. Papaemmanuil, P.J. Campbell, M. Cazzola, and R. Piazza Recurrent ETNK1 mutations in atypical chronic myeloid leukemia. Blood. 125: James, C., V. Ugo, J.P. Le Couedic, J. Staerk, F. Delhommeau, C. Lacout, L. Garcon, H. Raslova, R. Berger, A. Bennaceur-Griscelli, J.L. Villeval, S.N. Constantinescu, N. Casadevall, and W. Vainchenker A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 434: Jatiani, S.S., S.J. Baker, L.R. Silverman, and E.P. Reddy Jak/STAT pathways in cytokine signaling and myeloproliferative disorders: approaches for targeted therapies. Genes Cancer. 1: Klampfl, T., H. Gisslinger, A.S. Harutyunyan, H. Nivarthi, E. Rumi, J.D. Milosevic, N.C. Them, T. Berg, B. Gisslinger, D. Pietra, D. Chen, G.I. Vladimer, K. Bagienski, C. Milanesi, I.C. Casetti, E. Sant'Antonio, V. Ferretti, C. Elena, F. Schischlik, C. Cleary, M. Six, M. Schalling, A. Schonegger, C. Bock, L. Malcovati C. Pascutto, G. Superti-Furga, M. Cazzola, and R. Kralovics Somatic mutations of calreticulin in myeloproliferative neoplasms. The New England journal of Page 2 of 6 Printed: 5/3/2018 1:31:23 PM

3 medicine. 369: Kralovics, R., F. Passamonti, A.S. Buser, S.S. Teo, R. Tiedt, J.R. Passweg, A. Tichelli, M. Cazzola, and R.C. Skoda A gain-of-function mutation of JAK2 in myeloproliferative disorders. The New England journal of medicine. 352: Lasho, T.L., C.M. Finke, D. Zblewski, M. Patnaik, R.P. Ketterling, D. Chen, C.A. Hanson, A. Tefferi, and A. Pardanani Novel recurrent mutations in ethanolamine kinase 1 (ETNK1) gene in systemic mastocytosis with eosinophilia and chronic myelomonocytic leukemia. Blood cancer journal. 5:e Levine, R.L., M. Wadleigh, J. Cools, B.L. Ebert, G. Wernig, B.J. Huntly, T.J. Boggon, I. Wlodarska, J.J. Clark, S. Moore, J. Adelsperger, S. Koo, J.C. Lee, S. Gabriel, T. Mercher, A. D'Andrea, S. Frohling, K. Dohner, P. Marynen, P. Vandenberghe, R.A. Mesa, A. Tefferi, J.D. Griffin, M.J. Eck, W.R. Sellers, M. Meyerson, T.R. Golub, S.J. Lee, and D.G. Gilliland Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer cell. 7: Palandri, F., R. Latagliata, N. Polverelli, A. Tieghi, M. Crugnola, B. Martino, M. Perricone, M. Breccia, E. Ottaviani, N. Testoni, F. Merli, F. Aversa, G. Alimena, M. Cavo, G. Martinelli, L. Catani, M. Baccarani, and N. Vianelli Mutations and long-term outcome of 217 young patients with essential thrombocythemia or early primary myelofibrosis. Leukemia. 29: Pietra, D., S. Li, A. Brisci, F. Passamonti, E. Rumi, A. Theocharides, M. Ferrari, H. Gisslinger, R. Kralovics, L. Cremonesi, R. Skoda, and M. Cazzola Somatic mutations of JAK2 exon 12 in patients with JAK2 (V617F)-negative myeloproliferative disorders. Blood. 111: Rotunno, G., C. Mannarelli, P. Guglielmelli, A. Pacilli, A. Pancrazzi, L. Pieri, T. Fanelli, A. Bosi, A.M. Vannucchi, and I. Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood. 123: Rumi, E., D. Pietra, V. Ferretti, T. Klampfl, A.S. Harutyunyan, J.D. Milosevic, N.C. Them, T. Berg, C. Elena, I.C. Casetti, C. Milanesi, E. Sant'antonio, M. Bellini, E. Fugazza, M.C. Renna, E. Boveri, C. Astori, C. Pascutto, R. Kralovics, M. Cazzola, and I. Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative. 2014a. JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood. 123: Rumi, E., D. Pietra, C. Pascutto, P. Guglielmelli, A. Martinez-Trillos, I. Casetti, D. Colomer, L. Pieri, M. Pratcorona, G. Rotunno, E. Sant'Antonio, M. Bellini, C. Cavalloni, C. Mannarelli, C. Milanesi, E. Boveri, V. Ferretti, C. Astori, V. Rosti, F. Cervantes, G. Barosi, A.M. Vannucchi, M. Cazzola, and I. Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative. 2014b. Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis. Blood. 124: Scott, L.M., W. Tong, R.L. Levine, M.A. Scott, P.A. Beer, M.R. Stratton, P.A. Futreal, W.N. Erber, M.F. McMullin, C.N. Harrison, A.J. Warren, D.G. Gilliland, H.F. Lodish, and A.R. Green JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. The New England journal of medicine. 356: Low coverage regions: This list contains exons where the average sequencing depth (number of times a particular position is sequenced) is below our stringent cutoff of 300. The sequencing reads from these exons were manually reviewed. If high quality variants are detected in these regions they will be listed above in Tier 1 or Tier 2. NONE Page 3 of 6 Printed: 5/3/2018 1:31:23 PM

4 This result has been reviewed and approved by Jay Patel, M.D. BACKGROUND INFORMATION: Myeloid Malignancies Mutation Panel by Next Generation Sequencing or Myeloid Malignancies Somatic Mutation and Copy Number Analysis Panel CHARACTERISTICS: Myeloid malignancies are clonal disorders of hematopoietic stem and progenitor cells that include myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). Recent studies have identified a number of recurrently mutated genes with diagnostic and/or prognostic impact in myeloid malignancies. The presence of certain somatic variants may also guide treatment selection. This targeted variant panel by massively parallel sequencing (next generation sequencing) is a more cost-effective approach when compared to the cost of multiple single gene tests. This test can be used to complement the morphologic and cytogenetic workup of myeloid malignancies. GENES TESTED: ASXL1, ASXL2, BCOR, BCORL1, BRAF, BRINP3, CALR, CBL, CEBPA, CSF3R, DNMT1, DNMT3A, EED, ELANE, ETNK1, ETV6, EZH2, FLT3, GATA1, GATA2, HNRNPK, IDH1, IDH2, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, LUC7L2, MAP2K1, MPL, NOTCH1, NPM1, NRAS, NSD1, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RUNX1, SETBP1, SF1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, SUZ12, TET2, TP53, U2AF1, U2AF2, WT1, ZRSR2. A full list of the targeted regions of the above genes is available through this link: METHODOLOGY: Genomic DNA is isolated from peripheral blood or bone marrow and then enriched for the targeted exonic regions of the tested genes. The variant status of the 57 targeted genes is determined by massively parallel sequencing. The hg19 (GRCh37) human genome assembly is used as a reference for identifying genetic variants. LIMITATIONS: Variants outside the targeted regions or below the limit of detection will not be identified. In some cases, variants may not be identified due to technical limitations in the presence of pseudogenes or in repetitive or homologous regions. It is also possible some insertion/deletion variants may not be identified. The lower limit of detection for medium and large variants (greater than 30 bp) has not been validated. LIMIT OF DETECTION: 5 percent mutant allele for single nucleotide variants (SNV) and small variants (less than 30bp) ANALYTICAL SENSITIVITY (PPA): Analytical sensitivity for all variant classes available through this link: CLINICAL DISCLAIMER: Results of this test must always be interpreted within the context of clinical findings and other relevant data and should not be used alone for a diagnosis of malignancy. This test is not intended to detect minimal residual disease. See Compliance Statement B: aruplab.com/cs Cytogenomic Microarray SNP - Oncology Abnormal * (Ref Interval: Normal) CMA ONC TEST RESULT Page 4 of 6 Printed: 5/3/2018 1:31:23 PM

5 Clinically significant copy number changes and long contiguous stretches of homozygosity detected by SNP microarray analysis: Mosaic deletion within 7q22.1q22.3 Non-contiguous gains and losses on chromosome 20 Sex chromosome complement: XY (male) Interpretation: The microarray analysis showed evidence of deletions and duplications involving chromosome 20 in approximately 95% of this specimen. The signal pattern observed with probes on chromosome 20 could be due to a gain of a deleted chromosome 20, an isodicentric derivative chromosome 20, or another complex rearrangement. The microarray analysis does not provide information about chromosome structure. Correlation with other laboratory findings, especially with chromosome analysis if it was performed, is recommended. In addition this microarray analysis also showed a mosaic interstitial deletion within the long arm of chromosome 7 (approximately 7.3 Mb, from 7q22.1 to q22.3) in approximately 30% of this specimen. There are at least 132 genes within the deleted region, including CUX1. CUX1 is a tumor suppressor gene and haploinsufficiency of this gene have been associated with various myeloid neoplasms. Deletions involving chromosome 20 and the long arm of chromosome 7 are recurrent findings in de novo and treatment-related myeloid neoplasms, including myelofibrosis. Please correlate these results with clinical and other laboratory findings in this patient. No other significant DNA copy number changes or copy neutral long contiguous stretches of homozygosity were detected. Recommendation: If monitoring this clone is clinically indicated, follow up studies can be done with chromosome analysis and FISH with Del(20q) probe. Please note that the deletion detected on 7q is not targeted by the common Del(7q) probe, therefore monitoring for this abnormality can be done with microarray analysis. SUMMARY OF ABNORMALITIES DETECTED (PATHOGENIC, ACQUIRED): arr[hg19] 7q22.1q22.3(99,549, ,824,894)x1-2 arr[hg19] 20p13p11.1(61,568-25,739,440)x1 arr[hg19] 20p11.1q11.21(26,069,744-31,307,574)x3 arr[hg19] 20q11.21q13.13(31,307,678-49,591,274)x1 arr[hg19] 20q13.13q13.33(49,591,274-62,915,555)x3 References: 1) Brecqueville et al. Array comparative genomic hybridization and sequencing of 23 genes in 80 patients with myelofibrosis at chronic or acute phase. Haematologica 2014; 99(1): ) Song et al. Comparison of the Mutational Profiles of Primary Myelofibrosis, Polycythemia Vera, and Essential Thrombocytosis. Am J Clin Pathol 2017; 147(5): ) McNerney et al. CUX1 is a haploinsufficient tumor suppressor gene on chromosome 7 frequently inactivated in acute myeloid leukemia. Blood 2013; 121(6): ) Wong et al. Inactivating CUX1 mutations promote tumorigenesis. Nat Genet 2014; 46(1):33-8. Test Information: Chromosomal microarray analysis (CMA) was performed using Affymetrix CytoScan HD microarray. This microarray consists of 2,696,550 oligonucleotide probes across the genome, including 1,953,246 unique non-polymorphic probes, and 743,304 SNP (single nucleotide polymorphism) probes. Patient hybridization parameters are normalized to a reference set derived from 100 individuals with normal microarray results. Genomic linear Page 5 of 6 Printed: 5/3/2018 1:31:23 PM

6 positions are given relative to NCBI build 37 (hg19). Detected aberrations are reported when found to have clear or suspected clinical relevance; aberrations devoid of relevant gene content or reported as common findings in the general population may not be reported. This microarray and associated software (Chromosome Analysis Suite) are manufactured by Affymetrix and used by ARUP Laboratories for the purpose of identifying DNA copy number gains and losses associated with large chromosomal imbalances. This analysis will not detect all forms of polyploidy, balanced rearrangements (eg, inversions and balanced chromosomal translocations), small deletions, point mutations, and some mosaic conditions. While this assay has been extensively validated by ARUP Laboratories and other clinical laboratories per ACMG guidelines, it is not feasible to validate every potential genomic imbalance in the human genome. Furthermore, this technique only identifies the regions of imbalance; it does not provide information regarding the arrangement or mechanisms responsible. For these reasons, we may recommend that some chromosomal microarray results be characterized by fluorescence in situ hybridization (FISH) or standard chromosome analysis. The functional resolution of this assay varies across different samples dependent upon the size of the abnormality, probe density, tumor content and quality of the DNA obtained. On average, the limit of detection will vary from less than 100 kilobases for samples with high tumor content (generally greater than 70 percent) to several megabases for samples with lower tumor content (25-35 percent). The limit of detection for loss of heterozygosity (LOH) is approximately 3 megabases. This result has been reviewed and approved by Reha Toydemir, MD, PhD, FACMG Electronic Signature INTERPRETIVE INFORMATION: Cytogenomic Microarray SNP - Oncology Test developed and characteristics determined by ARUP Laboratories. See Compliance Statement B: aruplab.com/cs VERIFIED/REPORTED DATES Procedure Accession Collected Received Verified/Reported Myeloid Malignancy Proposed Diagnosis /2/2018 3:21:00 PM 5/2/2018 3:22:35 PM 5/3/2018 8:05:00 AM Myeloid Malignancies Panel Specimen /2/2018 3:21:00 PM 5/2/2018 3:22:35 PM 5/3/2018 8:05:00 AM Myeloid Panel Summary /2/2018 3:21:00 PM 5/2/2018 3:22:35 PM 5/3/2018 8:05:00 AM Myeloid Malignancies Panel Interp /2/2018 3:21:00 PM 5/2/2018 3:22:35 PM 5/3/2018 8:05:00 AM Cytogenomic Microarray SNP - Oncology /2/2018 3:21:00 PM 5/2/2018 3:22:35 PM 5/3/2018 8:05:00 AM END OF CHART Page 6 of 6 Printed: 5/3/2018 1:31:23 PM