Somatic GATA2

mutation is associated

with immunodeficiency

and pulmonary alveolar

proteinosis in a patient

with myeloproliferative

neoplasm

Collin, M

Dickinson, R

Lipman, M

Lowe, D

Marafioti, T

Mitchell, C

Pocock, R

Sekhar, M

UCL Discovery

EXCEPTIONAL CASE REPORTCan somatic GATA2 mutation mimic germ line GATA2 mutation?Mallika Sekhar,1,2 Rachael Pocock,3 David Lowe,4 Christopher Mitchell,3 Teresa Marafioti,5 Rachel Dickinson,6 Matthew Collin,6,* andMarc Lipman7,8,*1Department of Haematology, Royal Free London National Health Service (NHS) Trust, London, United Kingdom; 2UCL Haematology, University College London, London,United Kingdom; 3Department of Haematology, North Middlesex Hospital NHS Trust, London, United Kingdom; 4Department of Immunology, Royal Free Hospital NHS Trust,London, United Kingdom; 5Department of Histopathology, University College London Hospital NHS Trust, London, United Kingdom; 6Human Dendritic Cell Laboratory,National Institute for Health Research Newcastle Biomedical Research Centre, Institute of Cellular Medicine, Newcastle University and Newcastle upon Tyne Hospitals,Newcastle upon Tyne, United Kingdom; 7Department of Respiratory Medicine, Royal Free London NHS Trust, London, United Kingdom; and 8UCL Respiratory Medicine,University College London, London, United KingdomKey Points• Somatic GATA2mutation is associatedwith immunodeficiencyand pulmonary alveolarproteinosis in a patientwith myeloproliferativeneoplasm.IntroductionGATA2 is a zinc-finger transcription factor integral to hemopoietic stem cell regulation, mononucleardevelopment, and alveolar macrophage activity.1 Heterozygous germ line GATA2 mutations areassociated with mycobacterial infection, monocytopenia, and deficiency of B/natural killer cells, andpulmonary alveolar proteinosis (PAP). GATA2 mutations confer a 90% lifetime risk of developingmyelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) with mutations detected in 7% to 15%of MDS in young adults and children.2,3 GATA2 mutations can be present in germ line or arisesomatically, and, in either case, heterozygous mutations can prove clinically consequential. The clinicalfeatures of GATA2 mutation are largely known through studies of germ line mutation, which may beinherited or arise de novo.2-6 Somatic mutations have been reported as drivers of myeloid neoplasia,including myeloproliferative neoplasms (MPN), and are well described in leukemia arising in the contextof bi-allelic CEBPA mutation.7 However, features such as mononuclear cytopenia, immunodeficiency,and pulmonary alveolar proteinosis (PAP) have not been reported.PAP is a rare syndrome characterized by the abnormal accumulation of surfactant in the alveoli andterminal airways resulting in respiratory failure.8 Neutralizing anti–granulocyte macrophage colony-stimulating factor (anti–GM-CSF) antibodies are detected in the majority of patients with primary PAP.Secondary PAP (sPAP), which is associated with hematologic malignancies (chiefly MDS/AML), resultsfrom intrinsic dysregulation of alveolar macrophages.9,10 Median survival is ,50% at 2 years, andmortality is mainly due to PAP itself. Twenty-five percent of sPAP patients have germ line GATA2mutations, and one-fifth of germ line GATA2-mutated individuals develop PAP.1,3,9,10We report the first case of a patient with proven acquired/somatic GATA2 mutation who developedtypical immunodeficiency features of germ line GATA2 mutation, including monocytopenia, probablemycobacterial infection, and PAP, on a background of MPN/MDS.All data and samples were collected after informed consent in accordance with the Declaration ofHelsinki.Case descriptionA 58-year-old woman was investigated for incidental detection of thrombocytosis in 2006. Blood resultsshowed hemoglobin 111 g/L, white blood cell count 9.6 3 109/L, platelets 727 3 109/L, and meancorpuscular volume 103.1 fL. A provisional diagnosis of essential thrombocythemia or MDS was madeafter exclusion of secondary thrombocytosis. In 2009, she underwent a bone marrow (BM) biopsy, and adiagnosis of MPN/MDS was confirmed on the basis of hypercellular marrow, dyserythropoiesis, andgrade 2/4 reticulin. She had normal hearing, no evidence of human papillomavirus papillomatosis,hypogammaglobulinemia, or lymphedema and remained well until 2012.Submitted 19 September 2017; accepted 18 March 2018. DOI 10.1182/bloodadvances.2017012617.*M.C. and M.L. contributed equally to this study.The full-text version of this article contains a data supplement.© 2018 by The American Society of Hematology904 24 APRIL 2018 x VOLUME 2, NUMBER 8900800700600500Hb (g/L)Platelets (x109/L)40030020010002006200720082009201020112012Jan-13Sep-13Jan-15Feb-15Mar-15Apr-15May-15Jun-15Jul-15Aug-15Sep-15504540353025WBC (x109/L)N (x109/L)Mo (x109/L)B (x109/L)201510502006200720082009201020112012Jan-13Sep-13Jan-15Feb-15Mar-15Apr-15May-15Jun-15Jul-15Aug-15Sep-152006 2007 2008 2009 2010 2011 2012 Jan-13 Sep-13 Jan-15 Feb-15 Mar-15 Apr-15 May-15 Jun-15 Jul-15 Aug-15 Sep-15BM n/a MPN/MDS MF MFLungs ?PTB LRTI LRTI PAPMonocyte count 0.6 0.7 0.6 0.5 0.5 0.1 0.0 0.0 0.2 1.3 0.0 2.0 0.6 1.0 2.0 0.4 0.0 1.6Splenomegaly 12.6 cm 15.5 cm present present 14 cm present present 17 cm present np np presentRuxolitinib/day 10 20 20 30 40 30 10 0 0 Figure 1. Blood and BM investigations and clinical course (2006-2015). Progress of hemoglobin (Hb) and platelet count (top). Progress of white blood cell count(WBC), neutrophils (N), monocytes (Mo), and blasts (B) in the blood (middle). Table listing diagnostic details, absolute monocyte counts, and clinical events (bottom). LRTI,lower respiratory tract infection; MF, myelofibrosis.24 APRIL 2018 x VOLUME 2, NUMBER 8 SOMATIC GATA2 MUTATION MIMICS GERM LINE MUTATION 905In December 2012, she developed night sweats and weight losswith persistent respiratory symptoms and radiologic findingsconsistent with mycobacterial disease. She was refractory tostandard antibiotics, and although bronchoalveolar lavage (BAL)was smear and culture negative, empirical antimycobacterialtherapy led to clinical and radiographic resolution. At this time,she developed a leukoerythroblastic blood film with circulatingblasts and intermittent monocytopenia (Figure 1). She lackedmutations of JAK2, CALR, and MPL genes and in 2013 wasdiagnosed with secondary myelofibrosis based on BM biopsyfindings.In October 2014, she had new chest infiltrates associated withrespiratory symptoms, responding to antibiotics. In January 2015,she developed worsening fatigue, splenomegaly, and blood countsi iiiiiivAPatient Years fromdiagnosis(source)Acquired mutations Germline / Clinical FeaturesGATA2p.390delKASXL1p.T822Nfs*11This case 1 (PB) 0% NT MPN/MDSMonocytopeniaProbable mycobacterial infectionPAP3 (BM) 4% 0%8 (BM) 31% 6%9 (PB) 39% 46%9 (LN) 36% 52%9 (LUNG) 4% 4%GATA2p.390delKASXL1p.R693XDickinson5Patient 14.II.2-18 (Fib) 0% NT Germline GATA2 p.S201*MDSMonocytopeniaPAP-18 (LCL) 0% 9%0 (PB) 44% 49%GATA2p.390delKRUNX1p.Q237EfsX335Wang8Family 1 #30 39% 37% Germline GATA2 p.C298LfsX86MDSMonocytopeniaMycobacterial infectionBFigure 2. Imaging and molecular investigations. (A) Serial computed tomography chest images (2007-2015). (i) June 2007: no respiratory symptoms. Normal imaging.(ii) January 2015: no respiratory symptoms but evidence of progressive MPN/MDS. Early interstitial changes are present. (iii) June 2015: cough and breathlessness.Progressive interstitial changes plus air space shadowing are present. (iv) September 2015: respiratory failure secondary to PAP. “Crazy paving” changes plus consolidationcharacteristic of PAP visible. (B) Comparison of somatically acquired c.1168_1170delAAG p.390delK GATA2 mutations. Three patients have been reported with somaticallyacquired p.390delK mutation in GATA2. Germ line mutations in GATA2 and other cooperating mutations in ASXL1 or RUNX1 were also present in two previous cases. Fib,fibroblast; LCL, lymphoid cell line; LN, lymph node; NT, not tested; PB, peripheral blood.906 SEKHAR et al 24 APRIL 2018 x VOLUME 2, NUMBER 8showing monocytopenia. Allogeneic stem cell transplant (SCT)was discussed and ruxolitinib commenced. Within 1 month, hersymptoms and blood counts improved. Over the next 2 months,the dose of ruxolitinib was increased; her blood counts fluctuated,but splenomegaly resolved in June 2015 (Figure 1).However, by then, she reported weight loss, worsening coughwith breathlessness, and dystrophic nails consistent with fungalinfection. Chest computed tomography revealed progressivechanges with interstitial thickening and air space shadowingthroughout both lungs, plus small volume necrotic mediastinallymphadenopathy (Figure 2A). Sputum, BAL, and endobronchialultrasound biopsies of her central lymphadenopathy were culturenegative; lymph node histology showed acute necrotic changes.BAL contained branching hyphae consistent with Aspergillusspecies. BAL galactomannan was positive at 1.58, and voricona-zole was commenced.Repeat BM biopsy showed myelofibrosis with hypolobated nucleiand 5% to 10% blasts. Hydroxycarbamide was substituted forruxolitinib; her pulmonary symptoms worsened despite anti-infectivetreatment, and a thoracoscopic surgical biopsy of the lung confirmeda diagnosis of PAP. Anti–GM-CSF antibodies were negative. Hersymptoms and radiology progressed in line with PAP, and she died inSeptember 2015 of respiratory failure.She was investigated for the presence of GATA2 mutation. BMDNA was extracted from paraffin blocks using the QIAamp DNAFFPE Tissue Kit (56404; QIAGEN) according to the manufacturer’sinstructions. Using Sanger sequencing, the patient was foundto harbor a mutation in exon 8 of GATA2 (c.1168_1170delAAG;p.390delK) and a mutation of ASXL1 (c.2464_2465insA;p.T822Nfs*11). The GATA2 mutation was predicted to be disease-causing using the MutationTaster prediction program6 (MutationSurveyor software version 4.0.8). On retrospective analysis of herprevious DNA samples and estimation of variant allele frequency fromelectrophoretograms, thisGATA2mutation was not detected in 2006,increased in abundance from 2009 to 2015, and was only found at 4%in lung tissue in 2015, suggesting that it was an acquired somaticmutation (Figure 2B; supplemental Figures). ASXL1 mutation wasobserved emerging at later time points, probably as a subclone.Results and discussionIn summary, we present details of an adult with MPN/MDS evolvingto myelofibrosis over 7 years. She acquired a somatic GATA2mutation 3 years after initial diagnosis. Three years later, shedeveloped possible mycobacterial infection and myelofibrosis. Fora further 2 years, she had intermittent respiratory symptoms withprogressive pulmonary infiltrates from 2014 onwards, and PAP waseventually diagnosed. The presence of PAP with negative GM-CSFantibodies led to the identification ofGATA2mutation in our patient.Somatic mutations in GATA2 occur rarely in MPN and othermyeloid neoplasms,9,11-13 with the exception of MDS/AML associ-ated with biallelic CEBPA mutations.7 Our patient had a tri-nucleotide deletion c.1168_1170delAAG; p390delK, identical tothat previously reported as a second somatic mutation in 2 otherpatients with coexisting germ line GATA2 mutations (Figure 2B). Inour previous patient (14.II.2),5 p390delK was confirmed as somaticby serial sampling and sequencing of fibroblast and LCL DNA. In theother reported patient, p390delK was proven to be biallelic with thegerm lineGATA2mutation by TA-cloning.12 In all three, cooperatingASXL1 or RUNX1 mutations were observed at similar variant allelefrequency to the somaticGATA2 variant. The present case remainsunique in that an isolated somaticGATA2mutation was responsiblefor the GATA2-related disease.Discrete episodes of pulmonary symptoms occurred from 3 yearsafter first detection of her GATA2 mutation, around the time oftransformation to myelofibrosis. The initial episode was consistentwith probable mycobacterial disease and resolved with specifictreatment of this. In June 2015, she developed possible Aspergilluslung infection, though we believe that PAP was responsible for herdeath. The pulmonary episodes through 2014 may have repre-sented the early stages of PAP.GATA2-associated PAP has a poorprognosis9 but can be successfully treated with SCT.10,14,15 Amultidisciplinary approach by hematologists, pulmonologists, andradiologists is recommended to achieve an early diagnosis ofsPAP, definitive treatment, and improved outcomes.The origin of alveolar/tissue macrophages and the relativecontribution of yolk sac versus hemopoietic stem cells in thedevelopment and maintenance of alveolar macrophages10 isunclear in humans. Certainly, alveolar macrophages persist aftermany years of monocytopenia in GATA2 mutation.16 Although thissuggests that they can be self-maintaining in the absence ofinflammation, their dysfunction in the case of somaticGATA2mutationand the successful response of PAP to SCT14,15 both indicate thatalveolar macrophages can arise from hemopoietic stem cells ifrequired. Our patient with PAP associated with a somatic mutationdemonstrates that somatic mutation in myeloid progenitors can lead todisruption of tissue/alveolar macrophage function.To conclude, the combination of monocytopenia, probable myco-bacterial infection, and PAP in the context of myeloid malignancy iswell documented in germ lineGATA2mutation,2-6 but in our patient,these conditions are united by a somatic mutation. To ourknowledge, this is the first report of somatic GATA2 mutation in amyeloid malignancy leading to fatal PAP. The case suggests thatthe immune and hematologic consequences of the somaticmutation can closely resemble those observed with the germ linemutation.AcknowledgmentsPolymerase chain reaction and Sanger sequencing of BM and bloodDNA was performed by the NHS Northern Molecular GeneticsService and Source Bioscience (https://www.sourcebioscience.com/services/genomics/sanger-sequencing-service/). Lung tissue for anal-ysis was kindly provided by Martin Young, Consultant Histopathologistand Cytopathologist, Royal Free London NHS Trust.AuthorshipContribution: M.S. was involved in the care of the patient, collectedand analyzed data, andwrote the paper; D.L. was involved in the careof the patient and analysis of the data andcontributed to thewriting ofthe paper; R.P. collected the data and wrote the initial draft of thepaper; C.M. was involved in the care of the patient and procurementof samples; T.M. reviewed all bonemarrow histology; R.D. undertookall molecular genetic analysis of samples; M.C. was involved inplanning and interpretation of molecular analysis, interpretation,discussion of clinical and laboratory data and writing of the paper;and M.L. was involved in the care of the patient, collection andanalysis of data and writing the paper.24 APRIL 2018 x VOLUME 2, NUMBER 8 SOMATIC GATA2 MUTATION MIMICS GERM LINE MUTATION 907Conflict-of-interest disclosure: M.S. has received researchfunding and honoraria from Novartis. The remaining authors declareno competing financial interests.Correspondence: Mallika Sekhar, Department of Haematology,Royal Free London NHS Trust, Pond St, London NW3 2QG, UnitedKingdom; e-mail: mallika.sekhar@nhs.net.References1. Collin M, Dickinson R, Bigley V. Haematopoietic and immune defects associated with GATA2 mutation. Br J Haematol. 2015;169(2):173-187.2. Wlodarski MW, Hirabayashi S, Pastor V, et al. Prevalence, clinical characteristics and prognosis of GATA2-related myelodysplastic syndromes (MDS) inchildren and adolescents. Blood. 2016;127(11):1387-1397.3. Spinner MA, Sanchez LA, Hsu AP, et al. GATA2 deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity. Blood. 2014;123(6):809-821.4. Hsu AP, Sampaio EP, Khan J, et al. Mutations in GATA2 are associated with the autosomal dominant and sporadic monocytopenia and mycobacterialinfection (MonoMAC) syndrome. Blood. 2011;118(10):2653-2655.5. Dickinson RE, Milne P, Jardine L, et al. The evolution of cellular deficiency in GATA2 mutation. Blood. 2014;123(6):863-874.6. Pasquet M, Bellanne´-Chantelot C, Tavitian S, et al. High frequency of GATA2 mutations in patients with mild chronic neutropenia evolving to MonoMacsyndrome, myelodysplasia, and acute myeloid leukemia. Blood. 2013;121(5):822-829.7. Fasan A, Eder C, Haferlach C, et al. GATA2mutations are frequent in intermediate-risk karyotype AML with biallelic CEBPAmutations and are associatedwith favorable prognosis. [letter] Leukemia. 2013;27(2):482-485.8. Suzuki T, Trapnell BC. Pulmonary alveolar proteinosis syndrome. Clin Chest Med. 2016;37(3):431-440.9. Ishii H, Seymour JF, Tazawa R, et al. Secondary pulmonary alveolar proteinosis complicating myelodysplastic syndrome results in worsening of prognosis:a retrospective cohort study in Japan. BMC Pulm Med. 2014;14(1):37.10. Griese M, Zarbock R, Costabel U, et al. GATA2 deficiency in children and adults with severe pulmonary alveolar proteinosis and hematologic disorders.BMC Pulm Med. 2015;15(1):87.11. Papaemmanuil E, Gerstung M, Malcovati L, et al; Chronic Myeloid Disorders Working Group of the International Cancer Genome Consortium. Clinicaland biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013;122(22):3616-3627, quiz 3699.12. Wang X, Muramatsu H, Okuno Y, et al. GATA2 and secondary mutations in familial myelodysplastic syndromes and pediatric myeloid malignancies[letter] Haematologica. 2015;100(10):e398-e401.13. Lundberg P, Karow A, Nienhold R, et al. Clonal evolution and clinical correlates of somatic mutations in myeloproliferative neoplasms. Blood. 2014;123(14):2220-2228.14. Grossman J, Cuellar-Rodriguez J, Gea-Banacloche J, et al. Nonmyeloablative allogeneic hematopoietic stem cell transplantation for GATA2 deficiency.Biol Blood Marrow Transplant. 2014;20(12):1940-1948.15. Tholouli E, Sturgess K, Dickinson RE, et al. In vivo T-depleted reduced intensity transplantation for GATA2-related immune dysfunction. Blood. 2018;131(12):1383-1387.16. Bigley V, Haniffa M, Doulatov S, et al. The human syndrome of dendritic cell, monocyte, B and NK lymphoid deficiency. J Exp Med. 2011;208(2):227-234.908 SEKHAR et al 24 APRIL 2018 x VOLUME 2, NUMBER 8

Can somatic GATA2 mutation mimic germ line GATA2 mutation?

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Can somatic GATA2 mutation mimic germ line GATA2 mutation?

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